Defensin-encoding nucleic acid molecules derived from nicotiana alata, uses therfor and transgenic plants comprising same

ABSTRACT

The present invention provides nucleic acid molecules derived from  Nicotiana alata , which encode defensin-like molecules. The present invention contemplates the use of such nucleic acid molecules in the generation of transcienic plants having resistance or at least reduced sensitivity to plant pests including insects, microorganisms, fungi and/or viruses. The transcienic plants provided by the present invention include monocotyledonous and dicotyledonous plants, and particularly include crop plants and ornamental flowering plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.60/267,271, filed Feb. 8, 2001.

FIELD OF INVENTION

The present invention provides genetic molecules encoding plant floraldefensin-like molecules and their use in generating transgenic plantshaving resistance or at least reduced sensitivity to plant pestsincluding insects, microorganisms, fungi and/or viruses. The presentinvention further provides for the use of floral-and seed-deriveddefensins in the generation of insect resistance in plants. The plantsmay be monocotyledonous or dicotyledonous plants and are in particular,crop plants and ornamental flowering plants. The genetic molecules arealso useful in generating recombinant defensin-like molecules for use inthe topical application of compositions to prevent or otherwise retardpest-infestation of plants. The floral defensin-like molecules orgenetic molecules encoding same of the present invention may be usedalone or in combination with other agents such as a proteinase inhibitorprecursor or a nucleic acid molecule encoding same or other molecules ortheir encoding nucleotide sequences.

BACKGROUND OF THE INVENTION

References to any prior art in this specification is not, and should notbe taken, as an acknowledgement or any form of suggestion that thisprior art forms part of the common general knowledge in Australia or anyother country.

The increasing sophistication of recombinant DNA techniques is greatlyfacilitating research and development in the agricultural industry. Thisis particularly the case in the horticultural area including the area ofcrop research. Of particular importance are the development of herbicideresistant plants and the development of pathogen resistant plants.

A number of approaches have been adopted to induce herbicide resistancein plants. For example, genes encoding enzymes, which deactivate orneutralize the active components of herbicides, have been expressed inplants. Whilst there has been some success in this approach, it is onecomponent of a multi-disciplined and multi-strategy approach tomaximizing yields of crops and products of crops and for maximizingreturns from other activities within the agricultural and horticulturalindustries.

One of the major difficulties facing the agricultural and horticulturalindustries is the control of insect and other pathogen infestation ofplants. Insects and other pathogens account for millions of tonnes oflost production on an annual basis. Although insecticides and otheranti-pathogenic chemical agents have been successfully employed, thereis a range of environmental and regulatory concerns with the continueduse of chemical agents to control plant pests. Furthermore, theincreasing use of chemical pesticides is providing selective pressurefor the emergence of resistance in populations of pests. There isclearly a need to further investigate alternative mechanisms of inducingresistance in plants to pathogens such as insects, microorganisms,fungi, arachnid and viruses.

A range of genetic measures has been adopted in test trials. Whilst somesuccess has been achieved, it is important for new and alternativegenetic approaches to be developed to combat the difficulties ofresistance.

One approach which has been suggested is the use of a group of proteinscollectively known as “defensins”. The defensins have previously beenknown as γ-thionins and are structurally distinct from the α- andβ-thionin families. Most defensins isolated and studied to date havebeen derived from seeds, especially those from Raphanus sativus andother members of the Brassicaceae family. Seed defensins are small (˜5kDa) basic, cysteine-rich proteins and many have anti-fungal activity.

Over the last few years several cDNA clones have been isolated from thefloral organs of solanaceous plants and Arabidopsis that encode proteinsthat are related to seed defensins. Unlike seed defensins, floraldefensins are produced from precursor proteins that have an acidicC-terminal domain in addition to the defensin domain. The role of thisacidic domain is unknown.

The defensin domain has little sequence in common with seed-deriveddefensins apart from eight cysteine residues that are stronglyconserved. Although several cDNAs, which encode floral defensins, havebeen isolated, the corresponding proteins have not been isolated andtheir biological function has not been examined. The inventors haveisolated both the cDNA and the corresponding floral defensin from theornamental tobacco Nicotiana alata. They have determined that thedefensin precursor is processed proteolytically to release maturedefensin from the acidic C-terminal domain.

In accordance with the present invention, the inventors have determinedthat floral defensins have useful properties in inhibiting plant pestattack or infestation. Furthermore, a floral defensin from Nicotianaalata is shown to be particularly effective in controlling insectattack. The present invention also provides a new use of seed and knownfloral defensins in the control of insect infestation of plants.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word comprise, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1, <400>2, etc. A sequence listing isprovided after the claims.

The present invention relates generally to genetic molecules alone or incombination with other genetic molecules and their use to induceresistance in plants or parts of plants to pathogen infestation such asbut not limited to insect infestation. More particularly, the presentinvention provides genetic molecules encoding defensin-like moleculesalone or in combination with genetic molecules encoding a proteinaseinhibitor or precursor thereof or other active molecule to inhibitinsect, microbial, fungal, arachnid or viral attack or other form ofinfestation in plants. The present invention further encompassescompositions comprising the defensin-like molecules alone or incombination with a proteinase inhibitor or precursor thereof or otheractive molecule for topical application to plants or parts of plants toassist in the control of insect, microbial, fungal, arachnid or viralinfestation of plants. The present invention further contemplates theuse of the subject genetic molecules in the manufacture of transgenicplants with resistance or at least reduced susceptibility to insect,microbial, fungal, arachnid or viral attack or other form ofinfestation. The defensin molecules may also be used as molecularframeworks to carry heterologous amino acid sequences where the foldingof the molecule is altered to a more active form. The present inventionfurther encompasses genetic constructs comprising a promoter and/orother regulatory sequence naturally associated with the gene encodingthe defensin-like molecule. The promoter and/or other regulatorysequence may be operably linked to a cDNA molecule encoding thedefensin-like protein or may be operably linked to another gene ornucleotide sequence of interest such as but not limited to a geneencoding a proteinase inhibitor precursor. The present invention stillfurther extends to transgenic plants or parts of transgenic plants withresistance or at least reduced sensitivity to attack or other form ofinfestation by insects, microorganisms, fungi and/or viruses.Particularly preferred plants are food and non-food crops such as cottonplants.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide comprising, in itsprecursor form, an N-terminal signal domain, a mature domain and anacidic C-terminal domain wherein said polypeptide is produced duringflower development and its mature domain has activity against one ormore plant pests.

Another aspect of the present invention is directed to an isolatednucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide comprising, in itsprecursor form, an N-terminal signal domain, a mature domain and anacidic C-terminal domain wherein said polypeptide is produced duringflower development and its mature domain comprises the structure:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein the mature domainhas activity against one or more plant pests with the proviso that thepolypeptide is not FST or TPP3.

A further aspect of the present invention contemplates an isolatednucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide comprising, in itsprecursor form, an N-terminal signal domain, a mature domain and anacidic C-terminal domain wherein said polypeptide is produced in theepidermal layers of petals and sepals, the cortical cells of the styleand the connective tissue of the anthers and its mature domain comprisesthe structure:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein the mature domainhas activity against one or more plant pests with the proviso that thepolypeptide is not FST or TPP3.

Still another aspect of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide comprising, in itsprecursor form, an N-terminal signal domain, a mature domain and anacidic C-terminal domain wherein said mature domain comprises the aminoacid sequence set forth in SEQ ID NO:8 or an amino acid sequence havingat least about 70% similarity thereto or is encoded by a nucleotidesequence set forth in SEQ ID NO:7 or a nucleotide sequence having atleast about 70% similarity thereto or a nucleotide sequence capable ofhybridizing to SEQ ID NO:7 or its complementary form under lowstringency conditions at 42° C.

Still a further aspect of the present invention provides a geneticconstruct comprising a promoter or functional equivalent thereofoperably linked to a nucleotide sequence encoding a floral-derived,defensin-like molecule having a mature domain comprising the amino acidsequence:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein said mature domainexhibits inhibitory activity against plant pests such as insect pestswith the proviso that the defensin-like molecule is not FST or TPP3.

Yet another aspect of the present invention is directed to the aminoacid sequence of the mature domain comprising the amino acid sequence[SEQ ID NO:58]:

X₃₀X₃₁CX₃₂X₃₃X₃₄SX₃₅X₃₆FX₃₇GX₃₈CX₃₉X₄₀X₄₁X₄₂X₄₃CX₄₄X₄₅X₄₆CX₄₇X₄₈EX₄₉FX₅₀X₅₁GX₅₂CX₅₃X₅₄X₅₅X₅₆X₅₇X₅₈CX₅₉CTX₆₀X₆₁C wherein X₃₀ = R or Q X₃₁= E, I or T X₃₂ = K or E X₃₃ = T, A or S X₃₄ = E, P or Q X₃₅ = N, Q or HX₃₆ = T or R X₃₇ = P, K or H X₃₈ = I, L, P or T X₃₉ = I, F, S or V X₄₀= T, M, R or S X₄₁ = K, D, E or A X₄₂ = P or S X₄₃ = P, S or N X₄₄ = Ror A X₄₅ = K, T, S or N X₄₆ = A, Y or V X₄₇ = I, L, Q or H X₄₈ = S, K, Tor N X₄₉ = K or G X₅₀ = T, S, I, or V X₅₁ = D or G X₅₂ = H, R, or N X₅₃= S, P or R X₅₄ = K, W, A or G X₅₅ = I, L or F X₅₆ = L, Q, P or R X₅₇= R or P X₅₈ = R or K X₅₉ = L or F X₆₀ = K, S or R X₆₁ = P, N or H

Yet a further aspect of the present invention is directed to the maturedomain-encoding sequence operably linked to a signal domain comprisingthe amino acid sequence [SEQ ID NO:59]:

MX₁X₂SX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈AX₂₉ wherein X₁ = A, G or K X₂ = R, N, or LX₃ = L, I or M X₄ = C, F or R X₅ = F or L X₆ = M, F or I X₇ = A or S X₈= F, T or A X₉ = A, L, V or F X₁₀ = I, V, L or F X₁₁ = L or I X₁₂ = A, Ior M X₁₃ = M, A or F X₁₄ = M or L X₁₅ = L or I X₁₆ = F or V X₁₇ = V, Tor L X₁₈ = A, T or S X₁₉ = Y or T X₂₀ = E or G X₂₁ = V or M X₂₂ = noamino acid or G X₂₃ = no amino acid or P X₂₄ = no amino acid, M or V X₂₅= no amino acid or T X₂₆ = no amino acid, I or S X₂₇ = no amino acid, Aor V X₂₈ = Q or E X₂₉ = no amino acid or Q

Even still another aspect of the present invention is directed to themature domain-encoding sequence operably linked to an acidic C-terminaldomain comprising the sequence [SEQ ID NO:60]:

X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₈₀X₈₁X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ X₉₅ wherein X₆₂ = noamino acid or V X₆₃ = no amino acid or F X₆₄ = no amino acid or D X₆₅= no amino acid or E or K X₆₆ = no amino acid or K or I X₆₇ = no aminoacid or M or S X₆₈ = no amino acid or T, I or S X₆₉ = no amino acid or Kor E X₇₀ = no amino acid or T or V X₇₁ = no amino acid or G or K X₇₂= no amino acid or A X₇₃ = no amino acid or E X₇₄ = no amino acid or Ior T X₇₅ = no amino acid or L X₇₆ = no amino acid or A, V or G X₇₇ = noamino acid or E X₇₈ = no amino acid or E X₇₉ = no amino acid or A X₈₀= no amino acid or K X₈₁ = no amino acid or T X₈₂ = no amino acid or LX₈₃ = no amino acid or A or S X₈₄ = no amino acid or A or E X₈₅ = noamino acid or A or V X₈₆ = no amino acid or L or V X₈₇ = no amino acidor L X₈₈ = no amino acid or E X₈₉ = no amino acid or E X₉₀ = no aminoacid or E X₉₁ = no amino acid or I X₉₂ = no amino acid or M X₉₃ = noamino acid or D or M X₉₄ = no amino acid or N or E

Even yet another aspect of the present invention is directed to thedefensin-like molecule comprising the sequence [SEQ ID NO:61]:

MX₁X₂SX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈AX₂₉X₃₀X₃₁CX₃₂X₃₃X₃₄SX₃₅X₃₆FX₃₇GX₃₈CX₃₉X₄₀X₄₁X₄₂X₄₃CX₄₄X₄₅X₄₆CX₄₇X₄₈EX₄₉FX₅₀X₅₁GX₅₂CX₅₃X₅₄X₅₅X₅₆X₅₇X₅₈CX₅₉CTX₆₀X₆₁CX₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀X₈₁X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ wherein X₁ = A, G or K X₂ = R, N, or L X₃ = L, I or M X₄= C, F or R X₅ = F or L X₆ = M, F or I X₇ = A or S X₈ = F, T or A X₉= A, L, V or F X₁₀ = I, V, L or F X₁₁ = L or I X₁₂ = A, I or M X₁₃ = M,A or F X₁₄ = M or L X₁₅ = L or I X₁₆ = F or V X₁₇ = V, T or L X₁₈ = A, Tor S X₁₉ = Y or T X₂₀ = E or G X₂₁ = V or M X₂₂ = no amino acid or G X₂₃= no amino acid or P X₂₄ = no amino acid, M or V X₂₅ = no amino acid orT X₂₆ = no amino acid, I or S X₂₇ = no amino acic or A or V X₂₈ = Q or EX₂₉ = no amino acid or Q X₃₀ = R or Q X₃₁ = E, I or T X₃₂ = K or E X₃₃= T, A or S X₃₄ = E, P or Q X₃₅ = N, Q or H X₃₆ = T or R X₃₇ = P, K or HX₃₈ = I, L, P or T X₃₉ = I, F, S or V X₄₀ = T, M, R or S X₄₁ = K, D, Eor A X₄₂ = P or S X₄₃ = P, S or N X₄₄ = R or A X₄₅ = K, T, S or N X₄₆= A, Y or V X₄₇ = I, L, Q or H X₄₈ = S, K, T or N X₄₉ = K or G X₅₀ = T,S, I, or V X₅₁ = D or G X₅₂ = H, R, or N X₅₃ = S, P or R X₅₄ = K, W, Aor G X₅₅ = I, L or F X₅₆ = L, Q, P or R X₅₇ = R or P X₅₈ = R or K X₅₉= L or F X₆₀ = K, S or R X₆₁ = P, N or H X₆₂ = no amino acid or V X₆₃= no amino acid or F X₆₄ = no amino acid or D X₆₅ = no amino acid or Eor K X₆₆ = no amino acid or K or I X₆₇ = no amino acid or M or S X₆₈= no amino acid or T, I or S X₆₉ = no amino acid or K or E X₇₀ = noamino acid or T or V X₇₁ = no amino acid or G or K X₇₂ = no amino acidor A X₇₃ = no amino acid or E X₇₄ = no amino acid or I or T X₇₅ = noamino acid or L X₇₆ = no amino acid or A, V or G X₇₇ = no amino acid orE X₇₈ = no amino acid or E X₇₉ = no amino acid or A X₈₀ = no amino acidor K X₈₁ = no amino acid or T X₈₂ = no amino acid or L X₈₃ = no aminoacid or A or S X₈₄ = no amino acid or A or E X₈₅ = no amino acid or A orV X₈₆ = no amino acid or L or V X₈₇ = no amino acid or L X₈₈ = no aminoacid or E X₈₉ = no amino acid or E X₉₀ = no amino acid or E X₉₁ = noamino acid or I X₉₂ = no amino acid or M X₉₃ = no amino acid or D or MX₉₄ = no amino acid or N or E

Another aspect of the present invention is directed to the geneticconstruct comprising a nucleotide sequence selected from SEQ ID NO:7,SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17 or a nucleotide sequencehaving at least 70% similarity to one or more of SEQ ID NO:8, SEQ IDNO:14, SEQ ID NO:16 and SEQ ID NO:18 or a nucleotide sequence capable ofhybridizing to SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17or a complementary form thereof.

A further aspect of the present invention provides a genetic constructfor use in generating insect-resistant transgenic plants, saidtransgenic plants producing a defensin or defensin-like moleculeselected from SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO: 18as well as SEQ ID NO:20 to SEQ ID NO:49 or an amino acid sequence havingat least 70% similarity to any one of SEQ ID NO:8, SEQ ID NO:14, SEQ IDNO:16 and SEQ ID NO:18.

Still another aspect of the present invention further contemplates amethod for generating a plant with increased or enhanced resistance to aplant pest, said method comprising introducing into the genome of aplant cell or genome of a group of plant cells a genetic constructcomprising a promoter or functional equivalent thereof operably linkedto a nucleotide sequence encoding a floral-derived, defensin-likemolecule having a mature domain comprising the amino acid sequence:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein said mature domainexhibits inhibitory activity against plant pests such as insect pestsand regenerating a plant from said cell or group of cells.

Still a further aspect of the present invention is directed to thedefensin-like molecule comprising a mature domain having the amino acidsequence [SEQ ID NO:58]:

X₃₀X₃₁CX₃₂X₃₃X₃₄SX₃₅X₃₆FX₃₇GX₃₈CX₃₉X₄₀X₄₁X₄₂X₄₃CX₄₄X₄₅X₄₆CX₄₇X₄₈EX₄₉FX₅₀X₅₁GX₅₂CX₅₃X₅₄X₅₅X₅₆RX₅₇CX₅₉CTX₆₀X₆₁C wherein X₃₀ = R or Q X₃₁ = E,I or T X₃₂ = K or E X₃₃ = T, A or S X₃₄ = E, P or Q X₃₅ = N, Q or H X₃₆= T or R X₃₇ = P, K or H X₃₈ = I, L, P or T X₃₉ = I, F, S or V X₄₀ = T,M, R or S X₄₁ = K, D, E or A X₄₂ = P or S X₄₃ = P, S or N X₄₄ = R or AX₄₅ = K, T, S or N X₄₆ = A, Y or V X₄₇ = I, L, Q or H X₄₈ = S, K, T or NX₄₉ = K or G X₅₀ = T, S, I, or V X₅₁ = D or G X₅₂ = H, R, or N X₅₃ = S,P or R X₅₄ = K, W, A or G X₅₅ = I, L or F X₅₆ = L, Q, P or R X₅₇ = R o PX₅₈ = R or K X₅₉ = L or F X₆₀ = K, S or R X₆₁ = P, N or H

Yet another aspect of the present invention provides a method forgenerating a plant with increased or enhanced resistance to an insect,said method comprising introducing into the genome of a plant cell orgenome of a group of plant cells a genetic construct comprising apromoter or functional equivalent thereof operably linked to anucleotide sequence encoding a defensin-like molecule having a maturedomain comprising the amino acid sequence:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein said mature domainexhibits inhibitory activity against plant pests such as insect pestsand regenerating a plant from said cell or group of cells.

Yet a further aspect of the present invention provides a transfected ortransformed cell, tissue or organ from a plant or a transformedmicrobial cell, said cell, tissue or organ comprising a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding a polypeptide comprising, in its precursor form,an N-terminal signal domain, a mature domain and an acidic C-terminaldomain wherein said polypeptide is produced during flower developmentand its mature domain has activity against one or more plant pests.

Even still another aspect of the present invention is directed to thenucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide comprising, in itsprecursor form, an N-terminal signal domain, a mature domain and anacidic C-terminal domain wherein said polypeptide is produced duringflower development and its mature domain comprises the structure:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆ _(C)_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein the mature domainhas activity against one or more plant pests.

Even yet another aspect of the present invention, insofar as it relatesto plants, further extends to progeny of the plants engineered toexpress the nucleic acid molecule encoding the defensin-like molecule ora variant or homologue thereof as well as vegetative, propagative andreproductive parts of the plants, such as flowers (including cut orsevered flowers), parts of plants, fibrous material from plants (forexample, cotton) and reproductive portions including cuttings, pollen,seeds and callus.

Another aspect of the present invention provides a genetically modifiedplant cell or multicellular plant or progeny thereof or parts of agenetically modified plant capable of producing a heterologousdefensin-like molecule as herein described wherein said transgenic plantis resistant or has reduced sensitivity to plant pests such as insects.

A further aspect of the present invention comprises one or more geneticconstructs alone or in combination comprising a first promoter operablylinked to a first nucleotide sequence wherein said first nucleotidesequence encodes a defensin-like molecule capable of inhibiting a plantpest such as an insect, said construct further comprising a secondpromoter operably linked to a second nucleotide sequence wherein saidsecond nucleotide sequence encodes a proteinase inhibitor or precursorthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation of the nucleotide sequence (SEQ ID NO:17) andthe predicted amino acid sequence (SEQ ID NO:18) of NaPdf1 (Nicotianaalata plant defensin 1), the cDNA encoding the floral defensin fromNicotiana alata. Only one strand with the polarity of the mRNA is shownand the nucleotides are numbered above. The amino acid sequence, shownin single letter code, is given below the nucleotide sequence and isnumbered beginning with 1 for the first amino acid of the matureprotein. The putative signal peptide is indicated by negative numbersand is underlined. The mature protein is boxed and arrows depict thepredicted cleavage sites of the signal peptide and the end of the matureprotein. The first stop codon is marked with an asterisk (*) and the twopolyadenylation sites are in bold. For further detail, refer to Example7.

FIG. 2 is a diagrammatic representation of an RNA gel blot analysis ofNaPdf1 expression in various tissues of Nicotiana alata. Total RNA wasisolated from anthers at stages I (5–10 mm buds), II (20–30 mm buds) andIII (50–70 mm buds) of development, from pollen grains, and from maturepistil, ovary, petal, leaf and root tissues of N. alata(self-incompatibility genotype, S₂S₂), as shown in panel A. Panel Bshows the same RNA samples following staining with ethidium bromide.

FIG. 3 is a representation of an autoradiograph showing in situlocalization of NaPdf1 RNA. (A) A transverse section of a 1 cm longflower bud, after hybridization with a ³⁵S-labelled NaPdf1 anti-senseRNA probe. Heavy labelling of the epidermal cells of the petal (Pe) andsepal (Se), the cortical cells of the pistil (Pi) and the connectivetissue of the anther (A) can be detected. (B) The same section as in A,under higher magnification. (C) A similar section as in B, afterhybridization with a ³⁵S-labelled NaPdf1 sense RNA probe. No labellingis seen in any of the cells of the pistil (Pi), anther (A), petal (Pe)or sepal (Se).

FIG. 4A is a representation showing bacterial expression of N-terminalhexahistidine-tagged pro-defensin (6H.NaproPdf1) encoded by the NaPdf1cDNA. Lane 1: total protein extracted 6 h post-induction with 1 mM IPTGin 1×SDS sample loading buffer. Lane 2: soluble proteins in the 8 M urealysate. Lane 3: induced protein purified by IMAC. The proteins wereseparated on a 15% w/v SDS-polyacrylamdile gel and were stained withCoomassie Blue. Molecular size markers are the Broad Range standardsfrom Bio-Rad. The induced ˜12 kDa protein (arrowed) was substantiallypure after immobilized metal affinity chromatography (IMAC).

FIG. 4B shows a reverse-phase HPLC chromatogram of a sample of the metalaffinity purified 6H.NaproPdf1 protein (IMAC from A), eluted asdescribed in Example 5.

FIG. 5 is a representation showing immunoblot analysis of plant extractswith the antibodies raised to the bacterially expressed pro-defensin(6H.NaproPdf1) encoded by the NaPdf1 cDNA clone. (A) A diagrammaticrepresentation of the five stages of developing. N. alata flowers. (B) Arepresentation of an immunoblot of buffer soluble proteins (60 μg) fromflowers at the stages of development shown in (A). Proteins wereseparated on a 15% w/v SDS-polyacrylamide gel prior to transfer tonitrocellulose (0.22 μm) and imunoblotting with antibodies (1:2500)raised against bacterially expressed 6H.NaproPdf1 (see FIG. 4). Theantibodies bound specifically to three proteins. The smallest is thepredicted size of mature defensin (˜5 kDa) while the two larger speciesare probably the precursor and a processing intermediate. For details,see Examples 4 and 5.

FIG. 6 shows the purification of mature N. alata defoensin from flowerbuds. (A) Reverse-phase HPLC on an Aquapore RP-300 C8 column (4.6 mm×100mm, Brownlee). Proteins were extracted from flower buds and partiallypurified by gel filtration chromatography (see Example 5) beforeRP-HPLC. Proteins were applied in 0.1% v/v TFA and eluted with 60% v/vacetonitrile in 0.089% v/v TFA (buffer B) according to the gradient0–100% buffer B over 40 min at a flow rate of 1 mL/min. Eluted proteinswere detected by absorbance at 215 nm. (Inset) Protein in Peak Aseparated by 15% w/v SDS-PAGE and immunoblotted with anti-6H.NaproPdf1antibodies (Example 4). (B) N-terminal sequencing and mass spectrometryconfirmed the identity of Peak A as the mature defensin domain encodedby the NaPdf1 cDNA clone. “x” corresponds to an unassigned amino acidthat is probably a cysteine as predicted from the cDNA sequence

FIG. 7 is a series of electron micrographs showing the location of theN. alata defensin in anthers and ovaries from 10 mm flower buds. (A)Overview of the anther showing the cells of the connective tissue withelectron dense deposits (arrowed) in the vacuole. (B) Immunogoldlocalization of the defensin in the cells of the connective tissue ofthe anther. The antibody bound to the electron dense deposits in thevacuole (v) and did not bind to the cytoplasm (ct) or the cell wall(cw). (C) Immunogold localization of the defensin in the cortical cellsof the ovary. The antibody bound specifically to electron dense depositsin the vacuole and no binding was observed in the cytoplasm or cellwalls.

FIG. 8 is a schematic representation of the precursor proteins predictedfrom cDNA clones that encode floral and seed defensins. (A) Some floraldefensins are produced as precursor proteins with three distinctdomains: an ER signal sequence (left section of the diagram), a centralbasic domain (middle of the diagram) and a C-terminal domain rich inacidic amino acids (right section of the diagram). The predicted sizesof each of these domains for the N. alata defensin are shown below thediagram. The mature floral defensin is released after proteolyticcleavage (arrowed). (B) cDNAs for seed derived defensins encode proteinswith an ER signal sequence and a basic defensin domain, but noC-terminal acidic domain.

FIG. 9 is an alignment of the amino acid sequence of NaPdf1 with thepredicted amino acid sequences encoded from five other flower-derivedcDNA clones, as follows:

FST Gu et al., Mol. Gen. Genet. 234: 89–96, 1992; (flower specificthionin): TPP3: Milligan and Gasser, Plant Mol. Biol. 28: 691–711, 1995;NTS13: Li and Gray, Plant Physiology 120: 633, 1999; PPT: Karunanandaaet al., Plant Mol. Biol., 26: 459–464, 1994; ATPIIIa: Yu et al., DirectSubmission, Accession No. S30578, 1999.

Some, but not all floral defensins have a C-terminal acidic domain of32–33 amino acids.

FIG. 10 is an alignment of the amino acid sequence of the mature domainof NaPdf1 with the amino acid sequences of the mature domain of othermembers of the plant defensin family. The N-terminal amino acid in theRs-AFP1, Rs-AFP2, M1, M2A and M2B sequence which is represented by “pQ”is a pyroglutamic acid. The sequences are derived from the followingsources:

FST: Gu et al. (1992; supra) (SEQ ID NO:25); TPP3: Milligan and Gasser(1995; supra) (SEQ ID NO:26); p322: Steikema et al., Plant Mol. Biol.11: 255–269, 1988 (SEQ ID NO:27); PPT: Karunanandaa et al. (1994; supra)(SEQ ID NO:28); SE60: Choi et al., Plant Physiology 101: 699–700, 1993;Choi et al., Mol. Gen. Genet. 246: 266–268, 1995 (SEQ ID NO:29); γ1-H:Mendez et al., Eur. J. Biochem. 194: 533–539, 1990 (SEQ ID NO:30); M2A,M1 and M2B: Neumann et al., Int. J. Protein & Peptide Research 47:437–446, 1996 (SEQ ID NO: 31, SEQ ID NO:35 and SEQ ID NO:36,respectively); Pth-St1: Moreno et al., Eur. J. Biochem. 223: 135–139,1995 (SEQ ID NO:32); Rs-AFP1 and Rs-AFP2: Terras et al., J. BiologicalChemistry 267: 15301–15309, 1992; Terras et al., FEBS Letters 316:233–240, 1993; Terras et al., Plant Cell 7: 573–588, 1995; and Fant etal., The solution structure by ¹H-NMR of Rs-AFP1, a plant antifungalprotein from radish seeds. In: LP Ingman, J Jokissaari, J Lounila (eds),Abstracts of the 12th European Experimental NMR Conference, p 247, 1994(SEQ ID NO:33 and SEQ ID NO:34, respectively); γ1-P: Collila et al.,FEBS Letters 270: 191–194, 1990 (SEQ ID NO:37); γ2-P: Collila et al.,(1990; supra) (SEQ ID NO:38); 10kDa: Ishibashi et al., Plant Mol. Biol.15: 59–64, 1990 (SEQ ID NO:39); SIα2, SIα3 and SIα1: Bloch andRichardson, FEBS Letters 279: 101–104, 1991 and Nitti et al., Eur. J.Biochem. 228: 250–256, 1995 (SEQ ID NO:40, SEQ ID NO:41 and SEQ IDNO:43, respectively); Dm-AMP2, Ah-AMP1, Osborn et al., FEBS Letters 368:257–262, Hs-AFP1, Dm-AMP1 and 1995 (SEQ ID NO:42, SEQ ID NO:45, Ct-AMP1:SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:49, respectively); pI230 andP139: Chiang and Hagwiger, Mol. Plant-Microbe Interact. 4: 324–331, 1991(SEQ ID NO:44 and SEQ ID NO:48, respectively); NeThio1 and NeThio2:Yamada et al., Plant Physiology 115: 314, 1997; (SEQ ID NO:50 and SEQ IDNO:51); and NpThio1: Komori et al., Plant Physiology 115: 314, 1997 (SEQID NO:52).

FIG. 11 shows growth inhibition curves of various agents againstBotrytis cinerea, as monitored by absorbance at 595 nm. Each treatmentwas performed in quadruplicate. Purified NaPdf1 protein at 20 μg/ml wasassayed. Water and ovalbumin (20 μg/ml) served as negative controls anda mixture of the antifungal proteins α- and β-purothionin (20 μg/ml) wasused as a positive control.

FIGS. 12A–12C show growth inhibition curves of various agents againstFusarium oxysporum f. sp. dianthi (12A) and F. oxysporum f. sp.vasinfectum (12B and 12C), as monitored by absorbance at 595 nm. Eachtreatment was performed in quadruplicate. Purified NaPdf1 protein at 20μg/ml (12A and 12B) and 10 μg/ml (12C) were assayed. Water and ovalbumin(20 μg/ml, 12A and 12B; 10 μg/ml, 12C) served as negative controls, anda mixture of the antifungal proteins α- and β-purothionin (20 μg/ml, 12Aand 12B; 10 μg/ml, 12C) was used as a positive control.

FIG. 13 is a schematic of plant transformation constructs pFL1 andpHEX3, used for the transformation of tobacco and cotton. Bothconstructs contain the N. alata defensin, NaPdf1, under the control ofthe CaMV35S promoter/terminator. The region designated “surB” codes forresistance to the herbicide glean, and that designated “nptII” codes forresistance against the antibiotic kanamycin.

FIG. 14 shows representations of protein blots indicating expression of(A) N. alata proteinase inhibitor (NaPI) protein and (B) NaPdf1 proteinin transgenic tobacco plants. In A, lane 1: 25 ng NaPI; lane 2: 100 ngNaPI; lane 3: pHEX3.4; lane 4: untransformed W38 and lane 5: pFL1/W19.In B, lane 1: pHEX3.4; lane 2: pFL1/W19; lane 3: untransformed W38 andlane 4: N. alata bud extract. (C) indicates expression of NaPdf1 proteinin a transgenic cotton plant. Lane 1: 25 ng purified NaPdf1; lane 2:plant CT28.14.1 (transformed with unrelated plasmid) and lane 3: plantCT35.9.1 (transformed with pHEX3).

FIGS. 15A–15D show growth curves for H. punctigera and H. armigera fedon transgenic N. tabacum leaves (lines pHEX3.4 and pFL1/W19) transformedwith the NaPdf1 gene and an untransformed W38 parent plant. (15A)Survival of H. punctigera larvae, measured between days 2 and 18, (15B)the average mean weight of H. punctigera larvae measured between days 7and 18, (15C) survival of H. armigera larvae measured between days 3 and23, (15D) the average mean weight of H. armigera larvae measured betweendays 6 and 23.

FIG. 16 shows the average mean weight of H. armigera larvae fed onartificial diet containing either 0.03% NaPdf1, 0.3% NaPdf1, 0.3% NAPIor casein in place of the test protein (control). Weight of larvae wasmeasured after 6, 9, 12 and 14 days of feeding.

FIG. 17 shows the average mean weight of H. armigera larvae fed ontransgenic cotton (lines CT35.9.4 and CT35.125.1) transformed withNaPdf1 and non-transformed parent Coker 315 at day 8.

Table 1 is a summary of amino acid and nucleotide sequence identifiers.

Table 2 Artificial diet ingredients used in the feeding trial of H.armigera

TABLE 1 SEQUENCE ID NO: DESCRIPTION SEQ ID NO:1 Primer FST1 SEQ ID NO:2Primer FST2 SEQ ID NO:3 Primer PDF1 SEQ 1D NO:4 Primer PDF2 SEQ ID NO:5Primer FLOR1 SEQ ID NO:6 Primer FLOR2 SEQ ID NO:7 cDNA encoding maturedomain (NaPdfl) SEQ ID NO:8 Amino acid sequence corresponding to SEQ IDNO:7 SEQ ID NO:9 cDNA encoding N-terminal domain (NaPdfl) SEQ ID NO:10Amino acid sequence corresponding to SEQ ID NO:9 SEQ ID NO:11 cDNAencoding C-terminal acidic tail (NaPdfl) SEQ ID NO:12 Amino acidsequence corresponding to SEQ ID NO:11) SEQ ID NO:13 cDNA encodingN-terminal + mature domain (NaPdfl) SEQ ID NO:14 Amino acid sequencecorresponding to SEQ ID NO:13 SEQ ID NO:15 cDNA encoding mature + acidicC-terminal domain (NaPdfl) SEQ ID NO:16 Amino acid sequencecorresponding to SEQ ID NO:15 SEQ ID NO:17 cDNA encoding NaPdfl SEQ IDNO:18 Amino acid sequence corresponding to SEQ ID NO:17 SEQ ID NO:19cDNA corresponding to 3′ end of NaPdfl SEQ ID NO:20 Amino acid sequenceof full FST SEQ ID NO:21 Amino acid sequence of full TPP3 SEQ ID NO:22Amino acid sequence of full NTS13 SEQ ID NO:23 Amino acid sequence offull PPT SEQ ID NO:24 Amino acid sequence of full ATPIIIa SEQ ID NO:25Amino acid sequence of mature domain of FST SEQ ID NO:26 Amino acidsequence of mature domain of TPP3 SEQ ID NO:27 Amino acid sequence ofmature domain of P322 SEQ ID NO:28 Amino acid sequence of mature domainof PPT SEQ ID NO:29 Amino acid sequence of mature domain of SE60 SEQ IDNO:30 Amino acid sequence of mature domain of γ1-H SEQ ID NO:31 Aminoacid sequence of mature domain of M2A SEQ ID NO:32 Amino acid sequenceof mature domain of PTH-St1 SEQ ID NO:33 Amino acid sequence of maturedomain of Rs-AFP1 SEQ ID NO:34 Amino acid sequence of mature domain ofRs-AFP2 SEQ ID NO:35 Amino acid sequence of mature domain of M1 SEQ IDNO:36 Amino acid sequence of mature domain of M2B SEQ ID NO:37 Aminoacid sequence of mature domain of γ1-P SEQ ID NO:38 Amino acid sequenceof mature domain of γ2-P SEQ ID NO:39 Amino acid sequence of maturedomain of 10 kDa SEQ ID NO:40 Amino acid sequence of mature domain ofSIα2 SEQ ID NO:41 Amino acid sequence of mature domain of SIα3 SEQ IDNO:42 Amino acid sequence of mature domain of Dm-AMP2 SEQ ID NO:43 Aminoacid sequence of mature domain of SIα1 SEQ ID NO:44 Amino acid sequenceof mature domain of P1230 SEQ ID NO:45 Amino acid sequence of maturedomain of Ah-AMP1 SEQ ID NO:46 Amino acid sequence of mature domain ofHs-AFP1 SEQ ID NO:47 Amino acid sequence of mature domain of Dm-AMP1 SEQID NO:48 Amino acid sequence of mature domain of P139 SEQ ID NO:49 Aminoacid sequence of mature domain of Ct-AMP1 SEQ ID NO:50 Amino acidsequence of mature domain of NeThio1 SEQ ID NO:51 Amino acid sequence ofmature domain of NeThio2 SEQ ID NO:52 Amino acid sequence of maturedomain of NpThio1 SEQ ID NO:53 Amino acid sequence of mature domain ofNTS13 SEQ ID NO:54 Amino acid sequence of mature domain of PPT SEQ IDNO:55 Amino acid sequence of mature domain of ATPIIIa SEQ ID NO:56 cDNAencoding mature domain of NaPI SEQ ID NO:57 Amino acid sequencecorresponding to SEQ ID NO:56 SEQ ID NO:58 Consensus sequence of maturedomain of defensin SEQ ID NO:59 Consensus sequence of internal domain ofdefensin SEQ ID NO:60 Consensus sequence of C-terminal domain ofdefensin SEQ ID NO:61 Consensus amino acid sequence of defensin

TABLE 2 Control 0.3% NaPdf1 0.03% NaPdf1 0.3% NaPI Reagent (10 g) (3 g)(3 g) (3 g) Powdered 300 mg 90 mg 90 mg 90 mg cotton leaf Yeast 200 mg60 mg 60 mg 60 mg Wheatgerm 240 mg 72 mg 72 mg 72 mg Ascorbic 320 mg 96mg 96 mg 96 mg acid Sorbic acid 8 mg 2.4 mg 2.4 mg 2.4 mg Paraben 16 mg4.8 mg 4.8 mg 4.8 mg Linseed oil 8 μl 2.4 μl 2.4 μl 2.4 μl Wheatgerm 16μl 4.8 μl 4.8 μl 4.8 μl oil Casein 26.5 mg — 7.155 mg — Inhibitor — 530μl 53 μl 600 μl protein (15 mg/ml) Distilled 1.66 ml — 444 μl water Theabove reagents were mixed and then added to melted agar Agar 320 mg 96mg 96 mg 96 mg Distilled 6 ml 1.77 ml 1.8 ml l.7 ml water Add and mixAmpicillin 14 μl 4.2 μl 4.2 μl 4.2 μl (200 mg/ml) Streptomycin 14 μl 4.2μl 4.2 μl 4.2 μl (200 mg/ml)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the determination of thebiological properties of floral-derived, defensin-like molecules fromplants and the elucidation of new properties in seed-derived defensinsand previously known floral-derived defensins. Importantly, a novelfloral-derived, defensin-like molecule is described which exhibitsactivity against plant pathogens and in particular plant pests such asinsects and fungi. Other defensin molecules are described which arecontemplated to exhibit anti-insect activity.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide comprising, in itsprecursor form, an N-terminal signal domain, a mature domain and anacidic C-terminal domain wherein said polypeptide is produced duringflower development and its mature domain has activity against one ormore plant pests.

This aspect of the present invention does not extend to the defensinsFST [flower specific thionin] (Gu et al., {1992; supra}) or TPP3(Milligan and Gasser, {1995; supra}).

Reference herein to a “polypeptide” includes reference to a peptide orprotein. Generally, the polypeptide comprises cysteine residues, thelocation of which is conserved within members of floral andnon-floral-derived defensin molecules. The location of the eightcysteine residues may be defined as follows:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral.

Accordingly, another aspect of the present invention is directed to anisolated nucleic acid molecule comprising a sequence of nucleotidesencoding or complementary to a sequence encoding a polypeptidecomprising, in its precursor form, an N-terminal signal domain, a maturedomain and an acidic C-terminal domain wherein said polypeptide isproduced during flower development and its mature domain comprises thestructure:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein the mature domainhas activity against one or more plant pests with the proviso that thepolypeptide is not FST or TPP3.

The term “isolated” means that the nucleic acid molecule has undergoneat least one step towards being isolated or concentrated or enrichedfrom a more complex solution or source. For example, the term “isolated”includes nucleic acid molecules concentrated or enriched from abiological or chemical sample by precipitation, centrifigation,electrophoresis, micro-filtration, electroporation or chromatography.The term “isolated”, however, is in no way intended to limit the nucleicacid molecule to a particular location or state and the presentinvention extends to the nucleic acid molecule when introduced into thegenome of a cell or when it is resident in progeny of cells into whichthe nucleic acid molecule has been introduced into its genome.

Reference herein to a “nucleic acid molecule” includes reference to DNAor RNA (e.g. mRNA) or DNA/RNA hybrids. A nucleic acid molecule may beregarded inter alia as a genetic molecule, nucleotide sequence orpolynucleotide sequence. Preferably, the nucleic acid molecule is a cDNAmolecule although the present invention extends to genomic forms of thenucleic acid molecule. The nucleic acid molecule of the presentinvention may also encode separately the N-terminal signal domain, themature domain and/or the acidic C-terminal domain or combinationsthereof. For example, the nucleic acid molecule may encode for theN-terminal signal domain operably linked to the mature domain.Alternatively, it may encode for the mature domain operably linked tothe acidic C-terminal domain. The nucleic acid molecule may also encodeall three domains or comprise heterologous domains from other defensinor defensin-like molecules. The development of heterologousdefensin-like molecules is encompassed in the present invention andprovides a means of broadening the anti-insect or anti-pest spectrum. Aheterologous molecule may also comprise multiple mature domains andrandom repeats of mature domains or other domains required for activity.

Reference herein to production of the polypeptide during “flowerdevelopment” includes reference to production in flowering parts such asbut not limited to production in the pistils, anthers, ovaries, sepalsand petals of the flowering region. Preferably, the polypeptide isproduced in the epidermal layers of the petals and sepals, the corticalcells of the style and the connective tissue of the anthers.

Accordingly, another aspect of the present invention contemplates anisolated nucleic acid molecule comprising a sequence of nucleotidesencoding or complementary to a sequence encoding a polypeptidecomprising, in its precursor form, an N-terminal signal domain, a maturedomain and an acidic C-terminal domain wherein said polypeptide isproduced in the epidermal layers of petals and sepals, the corticalcells of the style and connective tissue of the anthers and its maturedomain comprises the structure:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein the mature domainhas activity against one or more plant pests with the proviso that thepolypeptide is not FST or TPP3.

In an alternative embodiment, the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide comprising two ormore mature domains having activity against one or more plant pests andoptionally an N-terminal signal domain and optionally an acidicC-terminal domain. The polypeptide according to the latter embodimentwould be regarded as a fusion polypeptide.

The location of production of the subject polypeptide may be altereddepending on the genetic construct employed to express the nucleic acidmolecule. For example, a developmentally regulated and/ortissue-specific promoter may be employed to direct expression of thenucleic acid molecule in any tissue. However, the naturally occurringpolypeptide is produced during flower development and more particularlyin the tissues of flowers outlined above.

The term “plant pests” is not to confer any limitation as to the type oforganism targeted by the subject defensin-like molecule. A plant pestincludes an insect, arachnid, microorganism, fungus or virus. In aparticularly preferred embodiment, the plant pest is an insect.

In a most preferred embodiment, the defensin-like molecule or itsencoding nucleic acid molecule is isolatable from N. alata and relatedspecies or varieties or strains thereof. The amino acid sequence of themature domain of the N. alata defensin is as follows (in single lettercode):

-   -   RECKTESNTF PGICITKPPC RKACISEKFT DGHCSKILRR CLCTKPC [SEQ ID        NO:8].

The nucleotide sequence encoding SEQ ID NO:8 is set forth in SEQ IDNO:7.

The present invention extends to novel variants of SEQ ID NO:8 such asvariants with an amino acid sequence having at least 70% similarity tothe sequence set forth in SEQ ID NO:8 or variants encoded by anucleotide sequence capable of hybridizing to the nucleotide sequenceencoding SEQ ID NO:8 (i.e. SEQ ID NO:7) under low stringency conditionsat 42° C.

Accordingly, another aspect of the present invention provides anisolated nucleic acid molecule comprising a sequence of nucleotidesencoding or complementary to a sequence encoding a polypeptidecomprising, in its precursor form, an. N-terminal signal domain, amature domain and an acidic C-terminal domain wherein said mature domaincomprises the amino acid sequence set forth in SEQ ID NO:8 or an aminoacid sequence having at least about 70% similarity thereto or is encodedby a nucleotide sequence set forth in SEQ ID NO:7 or a nucleotidesequence having at least about 70% similarity thereto or a nucleotidesequence capable of hybridizing to SEQ ID NO:7 or its complementary formunder low stringency conditions at 42° C.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In a particularly preferredembodiment, nucleotide and sequence comparisons are made at the level ofidentity rather than similarity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence similarity”, “sequence identity”,“percentage of sequence similarity, percentage of sequence identity”,“substantially similar” and “substantial identity”. A “referencesequence” is at least 12 but frequently 15 to 18 and often at least 25or above, such as 30 monomer units, inclusive of nucleotides and aminoacid residues, in length. Because two polynucleotides may each comprise(1) a sequence (i.e. only a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides., and (2) asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically 12 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e. gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by computerised implementations ofalgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science DriveMadison, Wis., USA) or by inspection and the best alignment (i.e.resulting in the highest percentage homology over the comparison window)generated by any of the various methods selected. Reference also may bemade to the BLAST family of programs as, for example, disclosed byAltschul et al. (Nucl. Acids Res. 25: 3389, 1997). A detailed discussionof sequence analysis can be found in Unit 19.3 of Ausubel et al.(“Current Protocols in Molecular Biology” John Wiley & Sons Inc,1994–1998, Chapter 15).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala,Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, H is, Asp,Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For the purposes of the present invention, “sequenceidentity” will be understood to mean the “match percentage” calculatedby the DNASIS computer program (Version 2.5 for windows; available fromHitachi Software engineering Co., Ltd., South San Francisco, Calif.,USA) using standard defaults as used in the reference manualaccompanying the software. Similar comments apply in relation tosequence similarity.

Reference herein to a “low stringency” includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is from about 25–30° C. to about 42° C. The temperaturemay be altered and higher temperatures used to replace formamide and/orto give alternative stringency conditions. Alternative stringencyconditions may be applied where necessary, such as medium stringency,which includes and encompasses from at least about 16% v/v to at leastabout 30% v/v formamide and from at least about 0.5 M to at least about0.9 M salt for hybridization, and at least about 0.5 M to at least about0.9 M salt for washing conditions, or high stringency, which includesand encompasses from at least about 31% v/v to at least about 50% v/vformamide and from at least about 0.01 M to at least about 0.15 M saltfor hybridization, and at least about 0.01 M to at least about 0.15 Msalt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C)% (Marmur and Doty, J. Mol. Biol. 5: 109, 1962).However, the T_(m) of a duplex DNA decreases by 1° C. with everyincrease of 1% in the number of mismatch base pairs (Bonner and Laskey,Eur. J. Biochem. 46: 83, 1974). Formamide is optional in thesehybridization conditions. Accordingly, particularly preferred levels ofstringency are defined as follows: low stringency is 6×SSC buffer, 0.1%w/v SDS at 25–42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/vSDS at a temperature in the range 20° C. to 65° C.; high stringency is0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.

The present invention is exemplified herein in relation to thedefensin-like molecules from N. alata. However, this is done with theunderstanding that the present invention extends to any novelfloral-derived, defensin-like molecule from any plant provided themolecule has activity against plant pests and in particular insects. Thepresent invention extends to derivatives of defensin-like moleculesincluding heterologous molecules as well as the use of known defensinsas anti-insect molecules.

Reference to a “defensin-like molecule” is made to highlight the factthat the present invention extends to homologues of defensin molecules.

The present invention further provides genetic constructs for use inexpressing defensin-like molecule-encoding nucleotide sequences inplants for the purposes of protecting the plant from plant pests.

Accordingly, another aspect of the present invention provides a geneticconstruct comprising a promoter or functional equivalent thereofoperably linked to a nucleotide sequence encoding a floral-derived,defensin-like molecule having a mature domain comprising the amino acidsequence:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein said mature domainexhibits inhibitory activity against plant pests such as insect pestswith the proviso that the defensin-like molecule is not FST or TPP3.

In a preferred embodiment, the amino acid sequence of the mature domaincomprises the amino acid sequence [SEQ ID NO:58]:

X₃₀X₃₁CX₃₂X₃₃X₃₄SX₃₅X₃₆FX₃₇GX₃₈CX₃₉X₄₀X₄₁X₄₂X₄₃CX₄₄X₄₅X₄₆CX₄₇X₄₈EX₄₉FX₅₀X₅₁GX₅₂CX₅₃X₄₃X₅₅X₅₆X₅₇X₅₈CX₅₉CTX₆₀X₆₁C wherein X₃₀ = R or Q X₃₁= E, I or T X₃₂ = K or E X₃₃ = T, A or S X₃₄ = E, P or Q X₃₅ = N, Q or HX₃₆ = T or R X₃₇ = P, K or H X₃₈ = I, L, P or T X₃₉ = I, F, S or V X₄₀= T, M, R or S X₄₁ = K, D, E or A X₄₂ = P or S X₄₃ = P, S or N X₄₄ = Ror A X₄₅ = K, T, S or N X₄₆ = A, Y or V X₄₇ = I, L, Q or H X₄₈ = S, K, Tor N X₄₉ = K or G X₅₀ = T, S, I, or V X₅₁ = D or G X₅₂ = H, R, or N X₅₃= S, P or R X₅₄ = K, W, A or G X₅₅ = I, L or F X₅₆ = L, Q, P or R X₅₇= R or P X₅₈ = R or K X₅₉ = L or F X₆₀ = K, S or R X₆₁ = P, N or H

The mature domain-encoding sequence may be operably linked to a signaldomain comprising the amino acid sequence [SEQ ID NO:59]:

MX₁X₂SX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈AX₂₉ wherein X₁ = A, G or K X₂ = R, N, or LX₃ = L, I or M X₄ = C, F or R X₅ = F or L X₆ = M, F or I X₇ = A or S X₈= F, T or A X₉ = A, L, V or F X₁₀ = I, V, L or F X₁₁ = L or I X₁₂ = A, Ior M X₁₃ = M, A or F X₁₄ = M or L X₁₅ = L or I X₁₆ = F or V X₁₇ = V, Tor L X₁₈ = A, T or S X₁₉ = Y or T X₂₀ = E or G X₂₁ = V or M X₂₂ = noamino acid or G X₂₃ = no amino acid or P X₂₄ = no amino acid, M or V X₂₅= no amino acid or T X₂₆ = no amino acid or S X₂₇ = no amino acid, A orV X₂₈ = Q or E X₂₉ = no amino acid or Q

In some cases, the mature domain-encoding sequence may be operablylinked to an acidic C-terminal domain comprising the sequence [SEQ IDNO:60]:

X₆₂X₆₃X₆₂X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀X₈₁X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ wherein X₆₂ = no amino acid or V X₆₃ = noamino acid or F X₆₄ = no amino acid or D X₆₅ = no amino acid or E or KX₆₆ = no amino acid or K or I X₆₇ = no amino acid or M or S X₆₈ = noamino acid or T, I or S X₆₉ = no amino acid or K or E X₇₀ = no aminoacid or T or V X₇₁ = no amino acid or G or K X₇₂ = no amino acid or AX₇₃ = no amino acid or E X₇₄ = no amino acid or I or T X₇₅ = no aminoacid or L X₇₆ = no amino acid or A, V or G X₇₇ = no amino acid or E X₇₈= no amino acid or E X₇₉ = no amino acid or A X₈₀ = no amino acid or KX₈₁ = no amino acid or T X₈₂ = no amino acid or L X₈₃ = no amino acid orA or S X₈₄ = no amino acid or A or E X₈₅ = no amino acid or A or V X₈₆= no amino acid or L or V X₈₇ = no amino acid or L X₈₈ = no amino acidor E X₈₉ = no amino acid or E X₉₀ = no amino acid or E X₉₁ = no aminoacid or I X₉₂ = no amino acid or M X₉₃ = no amino acid or D or M X₉₄= no amino acid or N or E

In yet another embodiment, the defensin-like molecule comprises thesequence [SEQ ID NO:61]:

MX₁X₂SX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂X₂₈AX₂₉X₃₀X₃₁CX₃₂X₃₃X₃₄SX₃₅X₃₆FX₃₇GX₃₈CX₃₉X₄₀X₄₁X₄₂X₄₃CX₄₄X₄₅X₄₆CX₄₇X₄₈EX₄₉FX₅₀X₅₁GX₅₂CX₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉CTX₆₀X₆₁CX₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ wherein X₁ = A, G or K X₂ = R, N, or L X₃ = L, I or M X₄= C, F or R X₅ = F or L X₆ = M, F or I X₇ = A or S X₈ = F, T or A X₉= A, L, V or F X₁₀ = I, V, L or F X₁₁ = L or I X₁₂ = A, I or M X₁₃ = M,A or F X₁₄ = M or L X₁₅ = L or I X₁₆ = F or V X₁₇ = V, T or L X₁₈ = A, Tor S X₁₉ = Y or T X₂₀ = E or G X₂₁ = V or M X₂₂ = no amino acid or G X₂₃= no amino acid or P X₂₄ = no amino acid, M or V X₂₅ = no amino acid orT X₂₆ = no amino acid, I or S X₂₇ = no amino acid or A or V X₂₈ = Q or EX₂₉ = no amino acid or Q X₃₀ = R or Q X₃₁ = E, I or T X₃₂ = K or E X₃₃= T, A or S X₃₄ = E, P or Q X₃₅ = N, Q or H X₃₆ = T or R X₃₇ = P, K or HX₃₈ = I, L, P or T X₃₉ = I, F, S or V X₄₀ = T, M, R or S X₄₁ = K, D, Eor A X₄₂ = P or S X₄₃ = P, S or N X₄₄ = R or A X₄₅ = K, T, S or N X₄₆= A, Y or V X₄₇ = I, L, Q or H X₄₈ = S, K, T or N X₄₉ = K or G X₅₀ = T,S, I, or V X₅₁ = D or G X₅₂ = H, R, or N X₅₃ = S, P or R X₅₄ = K, W, Aor G X₅₅ = I, L or F X₅₆ = L, Q, P or R X₅₇ = R or P X₅₈ = R or K X₅₉= L or F X₆₀ = K, S or R X₆₁ = P, N or H X₆₂ = no amino acid or V X₆₃= no amino acid or F X₆₄ = no amino acid or D X₆₅ = no amino acid or Eor K X₆₆ = no amino acid or K or I X₆₇ = no amino acid or M or S X₆₈= no amino acid or T, I or S X₆₉ = no amino acid or K or E X₇₀ = noamino acid or T or V X₇₁ = no amino acid or G or K X₇₂ = no amino acidor A X₇₃ = no amino acid or E X₇₄ = no amino acid or I or T X₇₅ = noamino acid or L X₇₆ = no amino acid or A, V or G X₇₇ = no amino acid orE X₇₈ = no amino acid or E X₇₉ = no amino acid or A X₈₀ = no amino acidor K X₈₁ = no amino acid or T X₈₂ = no amino acid or L X₈₃ = no aminoacid or A or S X₈₄ = no amino acid or A or E X₈₅ = no amino acid or A orV X₈₆ = no amino acid or L OR v X₈₇ = no amino acid or L X₈₈ = no aminoacid or E X₈₉ = no amino acid or E X₉₀ = no amino acid or E X₉₁ = noamino acid or I X₉₂ = no amino acid or M X₉₃ = no amino acid or D or MX₉₄ = no amino acid or N or E

In a preferred embodiment, the genetic construct comprises a nucleotidesequence encoding an amino acid sequence selected from SEQ ID NO:8, SEQID NO:14, SEQ ID NO:16 and SEQ ID NO:18.

In a particularly preferred embodiment, the genetic construct comprisesa nucleotide sequence selected from SEQ ID NO:7, SEQ ID NO:13, SEQ IDNO:15 and SEQ ID NO:17 or a nucleotide sequence having at least 70%similarity to one or more of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 andSEQ ID NO:18 or a nucleotide sequence capable of hybridizing to SEQ IDNO:7, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17 or a complementaryform thereof.

Most preferably, the amino acid sequence corresponds to the maturedomain and comprises the amino acid sequence set forth in SEQ ID NO:8.

Reference to SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18includes reference to novel variants having at least about 70%similarity to any one of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQID NO:18. These sequences may also be used to generate multimeric orheterologous molecules.

In accordance with this aspect of the present invention, the constructis for use in generating transgenic plants with increased or enhancedresistance to plant pests (e.g. insect), attack or infestation.Preferably, the construct is used solely for this purpose. The constructmay, however, be used for generating recombinant defensin-like moleculesin microorganisms such as bacteria. The present invention extends togeneric constructs encoding any defensin or defensin-like molecule foruse in combating insect infestation. For example, such constructs may beused to generate transgenic plants resistant to insects.

Accordingly, the present invention provides a genetic construct for usein generating insect-resistant transgenic plants, said transgenic plantsproducing a defensin or defensin-like molecule selected from SEQ IDNO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18 as well as SEQ IDNO:20 to SEQ ID NO:49 or an amino acid sequence having at least 70%similarity to any one of SEQ ID NO: 8, SEQ ID NO:14, SEQ ID NO:16 andSEQ ID NO:18.

As stated above, a plant includes either a monocotyledonous plant ordicotyledonous plant. Particularly, useful plants are food crops such aswheat, rice, barley, soybean and sugarcane. Particularly useful non-foodcommon crops include cotton. Flower and ornamental crops include rose,carnation, petunia, lisianthus, lily, iris, tulip, freesia, delphinium,limoniurn and pelargonium.

The present invention further contemplates a method for generating aplant with increased or enhanced resistance to a plant pest, said methodcomprising introducing into the genome of a plant cell or genome of agroup of plant cells a genetic construct comprising a promoter orfunctional equivalent thereof operably linked to a nucleotide sequenceencoding a floral-derived, defensin-like molecule having a mature domaincomprising the amino acid sequence:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein said mature domainexhibits inhibitory activity against plant pests such as insect pestsand regenerating a plant from said cell or group of cells. In oneaspect, this embodiment does not extend to defensin-like molecules FSTand TPP3.

The preferred plant pest is an insect.

Preferably, the defensin-like molecule comprises a mature domain havingthe amino acid sequence [SEQ ID NO:58]:

X₃₀X₃₁CX₃₂X₃₃X₃₄SX₃₅X₃₆FX₃₇GX₃₈CX₃₉X₄₀X₄₁X₄₂X₄₃CX₄₄X₄₅X₄₆CX₄₇X₄₈EX₄₉FX₅₀X₅₁GX₅₂CX₅₃X₅₄X₅₅X₅₆X₅₇X₅₈CX₅₉CTX₆₀X₆₁C wherein X₃₀ = R or Q X₃₁= E, I or T X₃₂ = K or E X₃₃ = T, A or S X₃₄ = E, P or Q X₃₅ = N, Q or HX₃₆ = T or R X₃₇ = P, K or H X₃₈ = I, L, P or T X₃₉ = I, F, S or V X₄₀= T, M, R or S X₄₁ = K, D, E or A X₄₂ = P or S X₄₃ = P, S or N X₄₄ = Ror A X₄₅ = K, T, S or N X₄₆ = A, Y or V X₄₇ = I, L, Q or H X₄₈ = S, K, Tor N X₄₉ = K or G X₅₀ = T, S, I, or V X₅₁ = D or G X₅₂ = H, R, or N X₅₃= S, P or R X₅₄ = K, W, A or G X₅₅ = I, L or F X₅₆ = L, Q, P or R X₅₇= R or P X₅₈ = R or K X₅₉ = L or F X₆₀ = K, S or R X₆₁ = P, N or H

More preferably, the mature domain is selected from the amino acidsequences set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 and SEQ ID NO:8.

Most preferably, the mature domain comprises an amino acid sequence setforth in SEQ ID NO:8.

Yet another aspect of the present invention provides a method forgenerating a plant with increased or enhanced resistance to an insect,said method comprising introducing into the genome of a plant cell orgenome of a group of plant cells a genetic construct comprising apromoter or functional equivalent thereof operably linked to anucleotide sequence encoding a defensin-like molecule having a maturedomain comprising the amino acid sequence:

a₁a₂C_(I)a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein said mature domainexhibits inhibitory activity against plant pests such as insect pestsand regenerating a plant from said cell or group of cells.

Preferred defensins are selected from SEQ ID NO:8, SEQ ID NO:18 and SEQID NO:20 to SEQ ID NO:49.

The mature domains referred to above include fragments and derivativesof these domains.

The term “fragment” as used herein means a portion or a part of themature domain parent which preferably retains the activity of the parentmature domain. The term “fragment” includes deletions, mutants and smallpeptides, for example, of at least 5, preferably at least about 10 andmore preferably at least about 20 contiguous amino acids, which comprisethe above anti-plant pest activity. Peptides of this type may beobtained through the application of standard recombinant nucleic acidtechniques or synthesized using conventional liquid or solid phasesynthesis as described, for example, in Chapter 9 entitled “PeptideSynthesis” by Atherton and Shephard which is included in a publicationentitled “Synthetic Vaccines” edited by Nicholson and published byBlackwell Scientific Publications. Alternatively, peptides can beproduced by digestion of an amino acid sequence of the invention withproteinases such as endoLys-C, endoArg-C, endoGlu-C and StaphylococcusV8-protease. The digested fragments can be purified, for example, byhigh performance liquid chromatographic (HPLC) techniques.

By “derivative” is meant a polypeptide that has been derived from thebasic sequence by modification, for example, by conjugation orcomplexing with other chemical moieties or by post-translationalmodification techniques as would be understood in the art. The term“derivative” also includes within its scope alterations that have beenmade to a parent sequence including additions or deletions that providefor functionally-equivalent molecules. Accordingly, the term“derivative” encompasses molecules that affect a plant's phenotype inthe same way as does the parent amino acid sequence from which it wasgenerated. Also encompassed are polypeptides in which one or more aminoacids have been replaced by different amino acids. It is well understoodin the art that some amino acids may be changed to others with broadlysimilar properties without changing the nature of the activity of thepolypeptide (conservative substitutions) as described hereinafter. Theseterms also encompass polypeptides in which one or more amino acids havebeen added or deleted or replaced with different amino acids.

The terms “protein”, “polypeptide”, “peptide” and “an amino acidsequence” are used interchangeably herein to refer to a polymer of aminoacid residues and to variants and synthetic analogues thereof. Thus,these terms apply to amino acid polymers in which one or more amino acidresidues is a synthetic non-naturally occurring amino acid, such as achemical analogue of a corresponding naturally occurring amino acid aswell as to naturally occurring amino acid polymers.

The terms “variant” and “homologue” refer to nucleotide sequencesdisplaying substantial sequence identity with reference nucleotidesequences or polynucleotides that hybridize with a reference sequenceunder stringency conditions that are herein defined. The terms “nucleicacid molecule”, “nucleotide sequence”, “polynucleotide” and “nucleicacid molecule” may be used herein interchangeably and encompasspolynucleotides in which one or more nucleotides have been added ordeleted or replaced with different nucleotides. In this regard, it iswell understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference nucleotide sequence whereby the altered polynucleotide retainsthe biological function or activity of the reference polynucleotide.Such variant polypeptide sequences may encode polypeptides comprisingsome differences in their amino acid composition but neverthelessencoding a protein having the same or similar activity. The resultingvariant polypeptide sequences are encompassed herein. The term “variant”also includes naturally occurring nucleotide allelic variants.

The term “expression” is used in its broadest sense and includestransient, semi-permanent and stable expression, as well as inducible,tissue-specific, constitutive and/or developmentally-regulatedexpression. Stable, tissue-specific expression is preferred.

To effect expression of the nucleotide sequence of the presentinvention, it may conveniently be incorporated into a chimeric geneticconstruct comprising inter alia one or more of the following: a promotersequence, a 5′ non-coding region, a cis-regulatory region such as afunctional binding site for transcriptional regulatory protein ortranslational regulatory protein, an upstream activator sequence, anenhancer element, a silencer element, a TATA box motif, a CCAAT boxmotif, an upstream open reading frame, transcriptional start site,translational start site, and/or nucleotide sequence which encodes aleader sequence, termination codon, translational stop site and a 3′non-translated region. Preferably, the chimeric genetic construct isdesigned for transformation of plants as hereinafter described.

The term “5′ non-coding region” is used herein in its broadest contextto include all nucleotide sequences which are derived from the upstreamregion of an expressible gene, other than those sequences which encodeamino acid residues which comprise the polypeptide product of said gene,wherein 5′ non-coding region confers or activates or otherwisefacilitates, at least in part, expression of the gene.

The term “gene” is used in its broadest context to include both agenomic DNA region corresponding to the gene as well as a cDNA sequencecorresponding to exons or a recombinant molecule engineered to encode afunctional form of a product.

As used herein, the term “cis-acting sequence” or “cis-regulatoryregion” or similar term shall be taken to mean any sequence ofnucleotides which is derived from an expressible genetic sequencewherein the expression of the first genetic sequence is regulated, atleast in part, by said sequence of nucleotides. Those skilled in the artwill be aware that a cis-regulatory region may be capable of activating,silencing, enhancing, repressing or otherwise altering the level ofexpression and/or cell-type-specificity and/or developmental specificityof any structural gene sequence.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences of a classicalgenomic gene, including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or environmental stimuli, or in a tissue-specific orcell-type-specific manner. A promoter is usually, but not necessarily,positioned upstream or 5′, of a structural gene, the expression of whichit regulates. Furthermore, the regulatory elements comprising a promoterare usually positioned within 2 kb of the start site of transcription ofthe gene.

In the present context, the term “promoter” is also used to describe asynthetic or fusion molecule, or derivative which confers, activates orenhances expression of a structural gene or other nucleic acid molecule,in a plant cell. Preferred promoters according to the invention maycontain additional copies of one or more specific regulatory elements tofurther enhance expression in a cell, and/or to alter the timing ofexpression of a structural gene to which it is operably connected.

The term “operably connected” or “operably linked” in the presentcontext means placing a structural gene under the regulatory control ofa promoter, which then controls the transcription and optionallytranslation of the gene. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition the genetic sequence or promoter at a distance from the genetranscription start site that is approximately the same as the distancebetween that genetic sequence or promoter and the gene it controls inits natural setting, i.e. the gene from which the genetic sequence orpromoter is derived. As is known in the art, some variation in thisdistance can be accommodated without loss of function. Similarly, thepreferred positioning of a regulatory sequence element with respect to aheterologous gene to be placed under its control is defined by thepositioning of the element in its natural setting, i.e. the genes fromwhich it is derived.

Promoter sequences contemplated by the present invention may be nativeto the host plant to be transformed or may be derived from analternative source, where the region is functional in the host plant.Other sources include the Agrobacterium T-DNA genes, such as thepromoters for the biosynthesis of nopaline, octapine, mannopine, orother opine promoters; promoters from plants, such as the ubiquitinpromoter; tissue specific promoters (see, e.g. U.S. Pat. No. 5,459,252to Conkling et al.; WO 91/13992 to Advanced Technologies); promotersfrom viruses (including host specific viruses), or partially or whollysynthetic promoters. Numerous promoters that are functional in mono- anddicotyledonous plants are well known in the art (see, for example,Greve, J. Mol. Appl. Genet. 1: 499–511, 1983; Salomon et al., EMBO J. 3:1984; Garfinkel et al., Cell 27: 143–513, 1983; Barker et al., PlantMol. Biol. 2: 235–350, 1983) including various promoters isolated fromplants (such as the Ubi promoter from the maize ubi-1 gene) (see, e.g.U.S. Pat. No. 4,962,028) and viruses (such as the cauliflower mosaicvirus promoter, CaMV 35S).

The promoter sequences may include regions which regulate transcription,where the regulation involves, for example, chemical or physicalrepression or induction (e.g. regulation based on metabolites, light, orother physicochemical factors; see, e.g. WO 93/06710 disclosing anematode responsive promoter) or regulation based on celldifferentiation (such as associated with leaves, roots, seed, or thelike in plants; see, e.g. U.S. Pat. No. 5,459,252 disclosing aroot-specific promoter). Thus, the promoter region, or the regulatoryportion of such region, is obtained from an appropriate gene that is soregulated. For example, the 1,5-ribulose bisphosphate carboxylase geneis light-induced and may be used for transcriptional initiation. Othergenes are known which are induced by stress, temperature, wounding,pathogen effects, etc.

The chimeric genetic construct of the present invention may alsocomprise a 3′ non-translated sequence. A 3′ non-translated sequencerefers to that portion of a gene comprising a DNA segment that containsa polyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. The polyadenylation signalis characterized by effecting the addition of polyadenylic acid tractsto the 3′ end of the mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon.

The 3′ non-translated regulatory DNA sequence preferably includes fromabout 50 to 1,000 nucleotide base pairs and may contain planttranscriptional and translational termination sequences in addition to apolyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. Examples of suitable 3′non-translated sequences are the 3′ transcribed non-translated regionscontaining a polyadenylation signal from the nopaline synthase (nos)gene of Agrobacterium tumefaciens (Bevan et al., Nucl. Acid Res. 11:369, 1983) and the terminator for the T7 transcript from the octopinesynthase gene of Agrobacterium tumefaciens. Alternatively, suitable 3′non-translated sequences may be derived from plant genes such as the 3′end of the protease inhibitor I or II genes from potato or tomato, thesoybean storage protein genes and the pea E9 small subunit of theribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene, although other3′ elements known to those of skill in the art can also be employed.Alternatively, 3′ non-translated regulatory sequences can be obtained denovo as, for example, described by An (Methods in Enzymology 153: 292,1987), which is incorporated herein by reference.

A chimeric genetic construct can also be introduced into a vector, suchas a plasmid. Plasmid vectors include additional DNA sequences thatprovide for easy selection, amplification and transformation of theexpression cassette in prokaryotic and eukaryotic cells, e.g.pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors,pSP-derived vectors, or pBS-derived vectors. Additional DNA sequencesinclude origins of replication to provide for autonomous replication ofthe vector, selectable marker genes, preferably encoding antibiotic orherbicide resistance, unique multiple cloning sites providing formultiple sites to insert DNA sequences or genes encoded in the chimericgenetic construct, and sequences that enhance transformation ofprokaryotic and eukaryotic cells.

The vector preferably contains an element(s) that permits either stableintegration of the vector or a chimeric genetic construct containedtherein into the host cell genome, or autonomous replication of thevector in the cell independent of the genome of the cell. The vector, ora construct contained therein, may be integrated into the host cellgenome when introduced into a host cell. For integration, the vector mayrely on a foreign or endogenous DNA sequence present therein or anyother element of the vector for stable integration of the vector intothe genome by homologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector or a constructcontained therein to be integrated into the host cell genome at aprecise location in the chromosome. To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to1,500 base pairs, preferably 400 to 1,500 base pairs, and mostpreferably 800 to 1,500 base pairs, which are highly homologous with thecorresponding target sequence to enhance the probability of homologousrecombination.

The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleic acidsequences.

For cloning and sub-cloning purposes, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously in ahost cell such as a bacterial cell. Examples of bacterial origins ofreplication are the origins of replication of plasmids pBR322, pUC19,pACYC177, and pACYC184 permitting replication in E. coli, and pUB110,pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. The originof replication may be one having a mutation to make its functiontemperature-sensitive in a Bacillus cell (see, e.g. Ehrlich, Proc. Natl.Acad. Sci. USA 75: 1433, 1978).

To facilitate identification of transformed cells, the vector desirablycomprises a further genetic construct comprising a selectable orscreenable marker gene. The actual choice of a marker is not crucial aslong as it is functional (i.e. selective) in combination with the plantcells of choice. The marker gene and the nucleotide sequence of interestdo not have to be linked, since co-transformation of unlinked genes as,for example, described in U.S. Pat. No. 4,399,216 is also an efficientprocess in plant transformation.

Included within the terms selectable or screenable marker genes aregenes that encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers that encode a secretable antigen that can be identifiedby antibody interaction, or secretable enzymes that can be detected bytheir catalytic activity. Secretable proteins include, but are notrestricted to, proteins that are inserted or trapped in the cell wall(e.g. proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S); small, diffusible proteinsdetectable, for example, by ELISA; and small active enzymes detectablein extracellular solution such as, for example, α-amylase, β-lactamase,phosphinothricin acetyltransferase).

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, erythromycin, chloramphenicolor tetracycline resistance. Exemplary selectable markers for selectionof plant transformants include, but are not limited to, a hyg gene whichencodes hygromycin B resistance; a neomycin phosphotransferase (neo)gene conferring resistance to kanamycin, paromomycin, G418 and the likeas, for example, described by Potrykus et al. (Mol. Gen. Genet, 199:183, 1985); a glutathione-S-transferase gene from rat liver conferringresistance to glutathione derived herbicides as, for example, describedin EP-A 256 223; a glutamine synthetase gene conferring, uponoverexpression, resistance to glutamine synthetase inhibitors such asphosphinothricin as, for example, described WO 87/05327, an acetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP-A 275 957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al. (Biotech. 6: 915, 1988), a bargene conferring resistance against bialaphos as, for example, describedin WO 91/02071; a nitrilase gene such as bxn from Klebsiella ozaenaewhich confers resistance to bromoxynil (Stalker et al., Science 242:419, 1988); a dihydrofolate reductase (DHFR) gene conferring resistanceto methotrexate (Thillet et al., J. Biol. Chem. 263: 12500, 1988); amutant acetolactate synthase gene (ALS), which confers resistance toimidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP-A-154204); a mutated anthranilate synthase gene that confers resistance to5-methyl tryptophan; or a dalapon dehalogenase gene that confersherbicide resistance.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known; a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known; an aequorin gene(Prasher et al., Biochem. Biophys. Res. Comm. 126: 1259, 1985), whichmay be employed in calcium-sensitive bioluminescence detection; a greenfluorescent protein gene (Niedz et al., Plant Cell Reports 14: 403,1995); a luciferase (luc) gene (Ow et al, Science 234: 856, 1986), whichallows for bioluminescence detection; a β-lactamase gene (Sutcliffe,Proc. Natl. Acad. Sci. USA 75: 3737, 1978), which encodes an enzyme forwhich various chromogenic substrates are known (e.g. PADAC, achromogenic cephalosporin); an R-locus gene, encoding a product thatregulates the production of anthocyanin pigments (red colour) in planttissues (Dellaporta et al., Chromosome Structure and Function, pp.263–282, 1988); an α-amylase gene (Ikuta et al., Biotech. 8: 241, 1990);a yrosinase gene (Katz et al., J. Gen. Microbiol. 129: 2703, 1983) whichencodes an enzyme capable of oxidizing tyrosine to dopa and dopaquinonewhich in turn condenses to form the easily detectable compound melanin;or a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA 80: 1101,1983), which encodes a catechol dioxygenase that can convert chromogeniccatechols.

The selectable marker gene construct may also comprise any one or moreof 5′ and 3′ non-coding regions, cis-regulatory regions, enhancers,activators and the like, as hereinbefore described.

A further aspect of the present invention provides a transfected ortransformed cell, tissue or organ from a plant or a transformedmicrobial cell, said cell, tissue or organ comprising a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding a polypeptide comprising, in its precursor form,an N-terminal signal domain, a mature domain and an acidic C-terminaldomain wherein said polypeptide is produced during flower developmentand its mature domain has activity against one or more plant pests.

Preferably, the nucleic acid molecule comprises a sequence ofnucleotides encoding or complementary to a sequence encoding apolypeptide comprising, in its precursor form, an N-terminal signaldomain, a mature domain and an acidic C-terminal domain wherein saidpolypeptide is produced during flower development and its mature domaincomprises the structure:

a₁a₂a₃a₄a₅a₆a₇a₈a₉a₁₀a₁₁a₁₂C_(II)a₁₃a₁₄a₁₅a₁₆a₁₇C_(III)a₁₈a₁₉a₂₀C_(IV)a₂₁a₂₂a₂₃a₂₄a₂₅a₂₆a₂₇a₂₈a₂₉C_(V)a₃₀a₃₁a₃₂a₃₃a₃₄a₃₅C_(VI)a₃₆C_(VII)a₃₇a₃₈a₃₉C_(VIII)wherein “a” may be the same or different and represents any amino acidresidue, the numerical subscript on each “a” represents its position inthe amino acid sequence and “C” represents a cysteine residue at aposition indicated by its Roman numeral and wherein the mature domainhas activity against one or more plant pests.

The vectors and chimeric genetic construct(s) of the present inventionmay be introduced into a cell by various techniques known to thoseskilled in the art. The technique used may vary depending on the knownsuccessful techniques for that particular organism.

Techniques for introducing vectors, chimeric genetic constructs and thelike into cells include, but are not limited to, transformation usingCaCl₂ and variations thereof, direct DNA uptake into protoplasts,PEG-mediated uptake to protoplasts, microparticle bombardment,electroporation, microinjection of DNA, microparticle bombardment oftissue explants or cells, vacuum-infiltration of tissue with nucleicacid, and T-DNA-mediated transfer from Agrobacterium to the planttissue.

For microparticle bombardment of cells, a microparticle is propelledinto a cell to produce a transformed cell. Any suitable ballistic celltransformation methodology and apparatus can be used in performing thepresent invention. Exemplary procedures are disclosed in Sanford andWolf (U.S. Pat. Nos. 4,945,050, 5,036,006, 5,100,792, 5,371,015). Whenusing ballistic transformation procedures, the genetic construct mayincorporate a plasmid capable of replicating in the cell to betransformed.

Examples of microparticles suitable for use in such systems include 0.1to 10 μm and more particularly 10.5 to 5 μm tungsten or gold spheres.The DNA construct may be deposited on the microparticle by any suitabletechnique, such as by precipitation.

Plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a chimericgenetic construct of the present invention and a whole plant generatedtherefrom. The particular tissue chosen will vary depending on theclonal propagation systems available for, and best suited to, theparticular species being transformed. Exemplary tissue targets includeleaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes,callus tissue, existing meristematic tissue (e.g. apical meristem,axillary buds, and root meristems), and induced meristem tissue (e.g.cotyledon meristem and hypocotyl meristem).

The regenerated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give a homozygous second generation (or T2) transformant and the T2plants further propagated through classical breeding techniques.

Accordingly, this aspect of the present invention, insofar as it relatesto plants, further extends to progeny of the plants engineered toexpress the nucleic acid molecule encoding the defensin-like molecule ora variant or homologue thereof as well as vegetative, propagative andreproductive parts of the plants, such as flowers (including cut orsevered flowers), parts of plants, fibrous material from plants (forexample, cotton) and reproductive portions including cuttings, pollen,seeds and callus.

Another aspect of the present invention provides a genetically modifiedplant cell Or multicellular plant or progeny thereof or parts of agenetically modified plant capable of producing a heterologousdefensin-like molecule as herein described wherein said transgenic plantis resistant or has reduced sensitivity to plant pests such as insects.

More particularly, the present invention provides a genetically modifiedplant cell or multi-cellular plant or progeny or parts thereofcomprising the amino acid sequence set forth in SEQ ID NO:8 or afragment or derivative.

The term “genetically modified” is used in its broadest sense andincludes introducing gene(s) into cells, mutating gene(s) in cells andaltering or modulating the regulation of gene(s) in cells.

The genetic construct may be a single molecule or multiple moleculessuch as a set of molecules such that a combination may comprisenucleotide sequences capable of encoding other anti-plant pathogenmolecules such as but not limited to a proteinase inhibitor or precursorthereof. Proteinase inhibitors such as serine proteinase inhibitorsfrequently accumulate in storage organs and in leaves in response towounding. The inhibitory activities of the proteins are directed againsta wide range of proteinases of microbial and animal origin.

Accordingly, another aspect of the present invention comprises one ormore genetic constructs alone or in combination comprising a firstpromoter operably linked to a first nucleotide sequence wherein saidfirst nucleotide sequence encodes a defensin-like molecule or partthereof capable of inhibiting a plant pest such as an insect, saidconstruct further comprising a second promoter operably linked to asecond nucleotide sequence wherein said second nucleotide sequenceencodes a proteinase inhibitor or precursor thereof.

In one embodiment, the defensin-like molecule and/or its encoding DNAsequence is selected from SEQ ID NO:7 to SEQ ID NO:18. In anotherembodiment, the defensin-like molecule and/or its encoding DNA sequenceis selected from SEQ ID NO:7 to SEQ ID NO:18 or SEQ ID NO:20 to SEQ IDNO:24. The defensin-like molecule and/or its encoding DNA sequencedefined by SEQ ID NO:7 or SEQ ID NO:8 is particularly preferred. In yetanother embodiment, the defensin-like molecule and/or its encoding DNAsequence is selected from SEQ ID NO:7 or SEQ ID NO:8, SEQ ID NO:17 orSEQ ID NO:18 or SEQ ID NO:20 to SEQ ID NO:55.

In another embodiment, the proteinase inhibitor is a serine proteinaseinhibitor of type I or type II. A particularly useful proteinaseinhibitor comprises the amino acid sequence encoding the nucleotidesequence set forth in SEQ ID NO:57 and SEQ ID NO:56, respectively. Thismolecule is also described in International Patent Application No.PCT/AU93/00659 (WO 94/13810).

The present invention extends to plants and plant parts includingreproductive parts of plants which carry all or part of a geneticconstruct which confers on the plant or plant parts resistance orreduced sensitivity to plant pests including insects and optionally asecond genetic construct as described above.

The present invention further contemplates a composition comprising adefensin or defensin-like molecule alone or in combination with anotheragent such as a proteinase inhibitor. The composition is particularlyuseful for topical application to plants such as cotton plants, toassist in controlling insect infestation.

The present invention further contemplates a promoter associated withthe genomic form of the nucleotide sequence set forth in SEQ ID NO:7.According to this aspect of the present invention, there is provided anisolated nucleic acid molecule having promoter activity andcorresponding to the genomic region in a plant genome which is operablylinked to a nucleotide sequence corresponding to all or part of SEQ IDNO:7 or a nucleotide sequence having 70% similarity thereof or anucleotide sequence capable of hybridizing to SEQ ID NO:7 or itscomplementary form.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Preparation of Plant Material

Nicotiana alata (Link et Otto) plants of mixed self-incompatibilitygenotype were maintained under glasshouse conditions as described byAnderson et al. (Plant Cell 1: 483–491, 1989). Flowers and floral budswere harvested and within two hours, pistils, ovaries and anthers wereremoved with forceps and a scalpel blade, while petals were separated byhand. Pollen grains from dehisced anthers and whole flowers at variousstages of development were also collected. The tissues were frozen inliquid nitrogen and stored at −70° C. until use.

EXAMPLE 2 Cloning of cDNA from N. alata

(a) Isolation of RNA

Total RNA was prepared by grinding 70 pistils (1.3 g) from N. alataflowers at the petal coloration stage of development to a fine powderwith a sterile mortar and pestle in the presence of liquid nitrogen. TheRNA was extracted with TRIzol (trademark) Reagent (Gibco BRL) accordingto the manufacturer's instructions (see Gibco BRL form #3796, TRIzol(trademark) Reagent Total RNA isolation reagent).

(b) cDNA Synthesis and Amplification of Floral Defensin Sequence by PCR

First strand cDNA was prepared from total pistil RNA using theSuperscript Preamplification System (Gibco BRL). Oligonucleotide primersused for PCR were specific to the DNA sequence published for the FlowerSpecific Thionin (FST, Gu et al. [1992; supra] from N. tabacum.

Primer FST1: 5′ GGAATTCCATATGGCTCGCTCCTTGTGC 3′ [SEQ ID NO:1] PrimerFST2 5′ GCGGATCCTCAGTTATCCATTATCTCTTC 3′ [SEQ ID NO:2]

Primer FST1 and primer FST2 matched the sequence of FST betweennucleotides 49–66, and 346–363 respectively. A cDNA clone encoding theN. alata floral defensin precursor (NaPdf1) was obtained by PCRamplification using the single-stranded cDNA as a template. The NaPdf1product was cloned into the pBluescript SK+ II (Stratagene) vector(pBS-NaPdf1) for sequencing. The product is 318 bp in length and encodesthe complete coding sequence without the 5′ and 3′ untranslated regions.

The NaPdf1 PCR product was subsequently used to screen a previouslyconstructed N. alata pistil cDNA library (Schultz et al., Plant Mot Biol35: 833–845, 1997). The membranes were probed with NaPdf1 cDNA labelledwith [α³²P]dCTP using random nonamer priming (Megaprime (trademark) DNAlabelling kit, Amersham Life Technologies). Unincorporated [α³²P]dCTPwas removed using a Bio-Rad Bio-Spin column 30 as described in themanufacturer's instructions. The blot was hybridized with the probe in asolution of 50% v/v formamide, 5×SSPE, 5×Denhardt's solution and 200μg/ml herring sperm DNA at 42° C. for 16 h before unbound probe wasremoved by washing twice with 2×SSPE and 0.1% w/v SDS at roomtemperature for 10 min. Hybridized probe was visualized by exposing theblot to x-ray film. Positive clones from the screen were excised andsequenced.

EXAMPLE 3 NaPdf1 Gene Expression

(a) RNA Gel Blot Analysis

Total RNA was isolated from anthers at stages I (5–10 mm buds), II(20–30 mm buds) and III (50–70 mm buds) of development, from pollengrains and from mature pistil, ovary, petal, leaf and root tissues of N.alata (self-incompatibility genotype, S₂S₂). The RNA samples (12.5 μg)were fractionated on a denaturing 1% w/v agarose-formaldehyde gel andtransferred to Hybond-N (Amersharn Life Sciences) membrane. Productionof a radio-labelled NaPdf1 cDNA probe and hybridization conditions wereas described for the screening of the cDNA library, in Example 2(b),above. Stringency washes were in 0.2×SSPE, 0.1% SDS at 45° C. and 55° C.for 30 min, respectively. Results are shown in FIG. 2. Hybridized probewas visualized by exposing the blot to a storage phosphor screen for 24h. The results were read in a Molecular Dynamics 400B phosphorimager,using the ImageQuant software.

(b) In Situ Hybridization

In situ hybridization was performed essentially as described by Drews etal. (Cell 65, 991–1002, 1991) and Cox and Goldberg (In: Analysis ofplant genes expression In Plant Molecular Biology: A Practical Approach.C. H. Shaw (ed), pp. 1–34, IRL Press, Oxford, United Kingdom, 1988).³⁵S-labelled sense and antisense RNA probes were produced by linearisingthe pBS-NaPdf1 DNA with EcoRI and BamHI and transcribing with T7 and T3RNA polymerases respectively (Ausubel et al. [1994; supra]; Drews et al.[1991; supra]) Ten mm flower buds were excised from the plant and fixedin 50% v/v ethanol, 5% v/v acetic acid and 3.7% v/v formaldehyde. Thefixed tissues were dehydrated and embedded in paraffin (Sigma). Thetissues were then sliced into 10 μm sections and attached toSuperfrost*/plus slides (Menzel-Glaser, Germany). The sections weretreated with xylene followed by hydration, proteinase K treatment,acetylation and dehydration. The ³⁵S-labelled sense and antisenseriboprobes were hydrolyzed to about 100 nucleotides in length andhybridized to the sections at 42° C. for 17 h. The sections were thentreated with ribonuclease A and washed, before the slides were coatedwith film emulsion. The sections were developed after a 1 week exposure.

Results indicated that NaPdf1 transcript accumulated in the connectivetissue of the anthers, the cortical cells of the style and in theepidermal cells of the petals and sepals (refer to FIG. 3). Thisaccumulation pattern is consistent with the encoded protein playing arole in the defense of the reproductive organs.

EXAMPLE 4 Protein Expression for Antibody Production

(a) Molecular Cloning and Bacterial Protein Expression

The pBS-NaPdf1 (see Example 2(b)) was used to PCR amplify a DNA fragmentencoding the proprotein domains (NaproPdf1, precursor minus theN-terminal ER signal domain). For a schematic representation of thestructure, see FIG. 8. The NaproPdf1 DNA fragment was obtained usingoligonucleotide primers PDF1 and PDF2 which incorporated a BamHI andSacI restriction site for subsequent cloning, respectively.

Primer PDF1 5′ CCGGATCCAGAGAATGCAAAACAG 3′ [SEQ ID NO:3] Primer PDF25′ GGGAGCTCTTAGTTATCCATTATCTC 3′ [SEQ ID NO:4]

The NaproPdf1 DNA fragment was cloned directly from PCR into the pGEM-Tvector (Promega) according to the manufacturer's instructions andsubcloned into the pQE30 (Qiagen) vector for protein expression in E.coli strain M15 bacteria (Qiagen).

The NaproPdf1 protein was expressed in bacteria, as a fusion with anN-terminal hexahistidine tag. Transformed E. coli cells were grown in LBbroth containing ampicillin (100 μg/ml) and kanamycin (12.5 μg/ml) to anabsorbance reading of 0.8 at 595 nm before induction with isopropylA-D-thiogalactopyranoside (IPTG, 1 mM) for 6 h. Cells were pelleted bycentrifugation and resuspended in lysis buffer (10 mM Tris-HCl pH 8.0,100 mM sodium phosphate buffer pH 8.0, 8 M urea; 30 ml of lysisbuffer/liter of culture) before incubation for 30 min on ice. The lysatewas then passed through an 18-gauge needle before the supernatant wascollected by centrifuigation (25,000 g, 15 min, 4° C.). Thehexahistidine-tagged proteins (6H.NaproPdf1) were purified from lysedbacterial cells using the denaturing protein purification protocoloutlined in the Clontech TALON (trademark) Metal Affinity Resin UserManual (PT1320–1). Bound proteins were eluted from the resin in 100 mMEDTA, pH 8.0. The eluted proteins were lyophilized. Protein extractscorresponding to various steps in the purification procedure wereanalyzed by SDS-PAGE (15% w/v polyacrylamide) and visualized by stainingwith Coomassie Blue (see FIG. 4A). A sample of the metal affinitypurified 6H.NaproPdf1 protein was applied to an Aquapore RP300reverse-phase C8 analytical column (4.6×100 mm, Brownlee) using a Watersmodel 510 pump and a Waters model 481UV detector. Protein was elutedwith a gradient of 0–100% buffer B (60% v/v acetonitrile In 0.089% v/vtrifluoroacetic acid), over 30 min. The protein eluted in a single peak(FIG. 4B) and N-terminal sequence and mass spectrometry confirmed thatit was 6H.NaproPdf1.

b) Production of a Polyclonal Antiserum

The bacterially-expressed 6H.NaproPdf1 protein (1.3 mg) was conjugatedto keyhole limpet hemocyanin (KLH, 0.3 mg, Sigma) with glutaraldehyde asdescribed by Harlow and Lane (Antibodies: A Laboratory Manual. ColdSprings Harbor Laboratory Press, Cold Springs Harbor, N.Y., 1988), priorto injection into a rabbit. The conjugated protein was then dialyzedagainst PBS overnight. The protein conjugate (100 μg, in 1 mL PBS) wasmixed with an equal volume of Freund's complete adjuvant (Sigma). Theprimary immunization consisted of 4×400 μL subcutaneous injections.Booster immunizations were administered 5 and 9 weeks later andcontained the protein conjugate (100 μg, in 1 mL PBS) mixed withFreund's incomplete adjuvant (Sigma). Pre-immune serum was collectedprior to injection while immune serum was collected 9 days after thesecond immunization.

(c) Protein A Purification of IgGs from Whole Sera

The IgG fraction in the pre-immune serum and immune serum were purifiedusing a Protein-A Sepharose CL-4B column (2.5 mL, Pharmacia) accordingto the manufacturer's instructions. The pre-immune serum (2 mL) wasdiluted in an equal volume of 0.1 M Tris-HCl (pH 7.5) and loaded ontothe column which had been equilibrated in 0.1 M Tris-HCl (pH 7.5). Thecluent was collected and re-applied three times. The column was washedwith 80 mL of 0.1 M Tris-HCl (pH 7.5), followed by 80 mL of 0.01 MTris-HCl (pH 7.5) to remove any unbound material. The IgGs were elutedwith 100 mM glycine (H 2.5) and 500 μL fractions were collected intomicrofaige tubes containing 50 μL of 1 M Tris-HCl (pH 8.8). Fractionscontaining the IgGs were pooled and dialysed extensively with PBS at 4°C. The Protein-A Sepharose column was regenerated by washing with 0.1 MTris-HCl (pH 7.5). When the pH reached 7.5, the immune serum (3 mL,second bleed) was applied and purified as described for the pre-immuneserum.

(d) SDS-Polyacrylamide Gel Electrophoresis

Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5 M NaCl) soluble proteinsamples (60 μg), from the stages of N. alata flower development shown inFIG. 5(A), were subjected to SDS-polyacrylamide gel electrophoresis(SDS-PAGE, with 4% w/v stacking and 15% resolving w/v polyacrylamidegels) (Laenumli, Nature 227: 680–685, 1970) using a Mini Protean IIElectrophoresis apparatus (Bio-Rad). The proteins were visualized bycoomassie blue staining and compared to Broad Range molecular weightmarkers (6.5–200 kDa) from Bio-Rad.

(e) Immunoblot Analysis

Proteins separated by SDS-PAGE were transferred to a nitrocellulosemembrane (Micron Separations Inc., 0.22 μm pore size) using aMini-Protean II Trans-Blot apparatus (Bio-Rad) in transfer buffer (48 mMTris (hydroxymethyl) aminomethane, 192 mM glycine, 20% v/v methanol).After transfer at 100 V for 1 h, the membrane was fixed in isopropanolfor 1 min followed by a 5 min wash in TBS (20 mM Tris-HCl, 150 mM NaCl,pH 8.0). Molecular weight markers were visualized by amido blackstaining (1:50 dilution of 0.1% w/v amido black in 40% v/v methanol and10% v/v acetic acid).

The membrane was blocked for 1 h in 5% w/v skim milk followed byincubation with anti-6H.NaproPdf1 antibodies for 1.5 h (1:2500 dilutionin TBS). The membrane was washed 3×10 min in TBST (0.1% v/v Tween 20 inTBS) before incubation with donkey anti-rabbit IgG conjugated tohorseradish peroxidase for 1.5 h (Amersham Pharmacia Biotech; 1:5000dilution in TBS). Three further 10 min washes were performed before theimmunoblot was treated with Enhanced Chemi-Luminescence (ECL) detectionreagents (Amersham Pharmacia Biotech) according to the manufacturer'sinstructions. Immunoblots were exposed to ECL Hyperfilm (AmershamPharmacia Biotech). Three proteins reacted specifically with theantibodies and the levels of protein were most abundant during the earlystages of development (refer to FIG. 5B).

The lower molecular weight species was confirmed as the mature defensinby mass spectrometry (5.3 kDa) and N-terminal amino acid sequencing;refer to Example 5(c), below.

EXAMPLE 5 Purification from Floral Buds

(a) Protein Extraction

Mature NaPdf1 protein was extracted from flowers using a modification ofa procedure for extraction of thionins from barley flour (Ozaki et al.,J. Biochem. 87: 549–555, 1980). Whole N. alata flowers up to the petalcoloration stage of flower development (5–50 mm, 650 g wet weight) wereground to a fine powder with liquid nitrogen using a mortar and pestleand processed further in an Ultra-Turrax homogenizer (Janke & Kunkel,Germany) in 50 mM sulfuric acid (3 mL/g weight). After stirring for 1 hat 4° C., insoluble material was removed by filtration through Miracloth(Calbiochem) followed by centrifugation (25,000×g, 15 min, 4° C.). Theslurry was adjusted to pH 7.8 by the slow addition of 10 M NaOH andstirred for 1 h at 4° C. before removal of precipitated material bycentrifugation (25,000×g, 15 min, 4° C.). Solid ammonium sulfate wasadded to 80% (w/v) saturation and the mixture was stirred for 4 h orovernight at 4° C. to precipitate the defensin protein. The precipitatewas collected by centrifiugation and was dissolved in 50 mL of gelfiltration buffer (150 mM KCl, 10 mM Tris-HCl, pH 8.0) prior to heatingat 90° C. for 10 min. Following centrifugation, the supernatant wasloaded onto a Sephadex G-50 gel filtration column (85×2.54 cm,Pharmacia). Fractions (50 mL) were collected and analyzed byimmnunoblotting with anti-6H.NaproPdf1 antibodies. Fractions containingNaPdf1 were pooled, concentrated by rotary evaporation at 45° C. andfiltered through a 0.22 μm syringe filter (Millipore) before furtherpurification by RP-EPLC.

(b) Reverse-phase High Performance Liquid Chromatography

RP-HPLC was performed on a Beckman System Gold HPLC coupled to aBeckmann 166 detector. Analytical RP-HPLC was conducted on an AquaporeRP300 reverse-phase C8 column (4.6×100 mm, Brownlee) while preparativeruns were performed using a Vydac C8 reverse-phase column (22×250 mm).The protein was eluted with a linear gradient of 0–100% buffer B (60%v/v acetonitrile in 0.089% v/v trifluoroacetic acid) at a flow-rate of 1mL/min or 10 mL/min over 40 min, respectively. Results from theanalytical column are shown in FIG. 6A. The identity of the major peakwas confirmed by N-terminal sequencing and mass spectrometry, asdescribed below.

(c) Electrospray Ionisation Mass Spectrometry

Prior to electrospray ionization mass spectrometry (ESI-MS), proteinfractions from RP-HPLC were concentrated under vacuum in a freeze drierand reconstituted in milli-Q water. ESI-MS was carried out using 1–100pmol protein in 2–4 μL of 50% v/v acetonitrile containing 0.1% w/vformic acid. Samples were infused at a flow rate of 0.2 μL/in into aPerkin-Elmer Sciex API-300 triple quadruple fitted with a micro-ionsprayion source. The mass scale was calibrated using singly-chargedpoly(propylene glycol) ions to a mass accuracy equivalent to ±1%. Massspectra were recorded in the first quadruple (Q1) scan mode over themass range m/z 200 to 3000 daltons per charge using a constant peakwidth (full width at half peak maximum) of 0.6 daltons per unit charge.Perkin-Elmer Sciex Bio-Multiview software was employed for signalaveraging of 30–100 scans, manual mass determination and transformationof mass-to-charge ratio spectra to a true mass scale. Uncertainties werecalculated at 95% confidence limits using small sample statistics andinclude calibration uncertainty.

Results indicated that the protein in Peak A in FIG. 6A, and the lowermolecular weight species shown in FIG. 5B (˜5 kDa), corresponded to themature defensin domain encoded by the NaPdf1 cDNA clone (see FIG. 6B).

(d) Amino Acid Sequencing

Amino acid sequencing by Edman degradation was carried out on an AppliedBiosystems 470A gas-phase peptide sequenator coupled to an AppliedBiosystems 130A separation system for automatic on-line PTH amino acidanalysis. The eight amino acids of N-terminal sequence obtained from theprotein in Peak A matched the sequence predicted from the NaPdf1 cDNAclone (see FIG. 6B).

EXAMPLE 6 Immunogold Localization

Fixation and Immunogold Labelling for Electron Microscopy

Anthers and ovaries were removed from N. alata flowers at the immaturebud stage (10 mm) and were fixed for 2 h at room temperature and thenovernight at 4° C. in 4% (w/v) formaldehyde and 0.5% (w/v)glutaraldehyde in 60 mM PIPES/KOH, pH 7.2. After fixation, the tissueswere washed in 60 mM PIPES/KOH, pH 7.2 and dehydrated for 3 h at roomtemperature in acidified dimethoxypropane (concentrated hydrochloricacid:dimethoxypropane, 1:2000 [v/v]). The dehydrated segments wereembedded in LR Gold containing Benzil (London Resin Co. Ltd.) bypolymerization under a Phillips TUV 15-W UV lamp at a distance of 10 cmfor 12 h at 25′,C. Immunogold labelling of ultrathin sections wasperformed as described in Anderson et al. (Planta 171: 438–442, 1987).The protein A purified anti-6H.NaproPdf1 antibodies were incubated withanther and ovary sections at a final concentration of 64 μg IgG/mL and21 μg IgG/mL, respectively. Specificity of labeling was tested byreplacing the primary antibody with antibodies purified from pre-immuneserum at the same concentration. For visualization of ultrastructure,the sections were stained for 15 min in 3% (w/v) aqueous uranyl acetateand 2 min with Sato triple lead stain (Sato, J. Electron Microse. 17:158–159, 1968) before being viewed on a Joel 1200 electron microscope.

EXAMPLE 7 Sequences and Sequence Comparisons

The nucleotide and predicted amino acid sequences of N. alata defensinare set forth in FIG. 1. The gene that the cDNA clone represents isdesignated NaPdf1 (N. alata plant defensin 1). The DNA sequence shown isa composite sequence from an overlapping cDNA clone and a primerextension product as follows: 1–49, primer extension product clone;50–541, cDNA clone NaPdf1. The protein predicted by this composite cDNAsequence is shown below the nucleotide sequence. The cDNA clone containsa single open reading frame of 318 nucleotides, encoding for 105 aminoacids. The PCR amplified sequence in the original pBS-NaPdf1 clone (seeExample 2) corresponds to nucleotides 1-318 in the NaPdf1 clone shown inFIG. 1.

In FIG. 9, the 105-amino acid sequence of NaPdf1 (SEQ ID NO:18) is shownaligned with the predicted amino acid sequences encoded by five otherflower-derived cDNA clones. The sequences, in order, are as follows:flower specific thionin (FST), sourced from Gu et al. [1992; supra] (SEQID NO: 20), derived from tobacco flowers; TPP3, sourced from Milliganand Gasser [1995; supra] (SEQ ID NO: 21), derived from tomato pistil;NTS13, sourced from Li and Gray [1999; supra] (SEQ ID NO: 22), derivedfrom tobacco styles; PPT, sourced from Karunanandaa et al. [1994; supra](SEQ ID NO: 23), derived from petunia pistil, and ATPIIIa, sourced fromYu et al. [1999; supra], Direct Submission, Accession No. S30578 (SEQ IDNO: 24), derived from Arabidopsis.

The 47 amino acids constituting the mature central domain of the NaPdf1protein (SEQ ID NO:8) were also aligned with the corresponding aminoacid sequences of the mature domains of other members of the plantdefensin family (SEQ ID NO:25 to SEQ ID NO:49). Alignment was carriedout using the computerized algorithm of ClustalW. The results are setforth in FIG. 10. For details of the relevant references from which eachsequence was obtained, and for their individual sequence identifiers,refer to the figure legend.

EXAMPLE 8 Fungal Growth Inhibition Assays

The 96-well microtitre plate assay of Broekaert et al. (EMS MicrobiologyLetters 69: 55–60, 1990) was used to test the effect of the purifiedNaPdf1 protein on the growth of Botrytis cinerea (isolated fromrosemary, Brunswick, Victoria), Fusarium oxysporum (f. sp. dianthi, Race2; isolated from carnation by Florigene Limited, Collingwood, Victoria)and Fusarium oxysporum (f. sp. vasinfectum, isolate VCG 01111 fromcotton; provided by the Department of Primary Industries, Queensland).Fungal spores were isolated from sporulating cultures growing on halfstrength potato dextrose agar (PDA, Difco) or gamma-irradiated carnationleaves in water agar by the addition of sterile water and the use of aspreader. The suspension was filtered through two layers of autoclavedmuslin and the spore concentration in the filtrate determined using ahaemocytometer. The spores were used directly or after storage insterile 20% v/v glycerol solution at −20° C.

Antifungal assays were performed in 96-well flat-bottomed microtitreplates (Greiner) under aseptic conditions. The spore concentration wasadjusted to about 2×10⁴ spores/mL in PDB and 80 mL of this suspensionwas added to each well to which 20 μL of filter sterilised (0.22 μmsyringe filter, Millipore) test protein (10, 50 or 100 μg/mL) or waterwas added. The purity and concentration of each protein was confirmedbefore use by RP-HPLC analysis. Sterile water and ovalbumin (Sigma) wereadded to other wells as negative controls, and a mixture of theantifungal proteins α- and β-purothionin (Sigma) was used as a positivecontrol. The plates were shaken on an orbital shaker for a few secondsto mix the spores and the test proteins. The plates were allowed tostand for 30 min to allow the spores to sediment before the opticaldensity of the plates were determined using a Spectra Max Pro 250microplate reader (Molecular Devices) at 595 nm absorbance. The plateswere incubated in the dark at 22° C. and measurements were taken overthe course of 100 h. An increase in absorbance relative to the initialreading was correlated with the growth of fungal hyphae and this wasplotted against time. All assays were performed in quadruplicate.

Growth inhibition curves, set out in FIGS. 11 and 12A–12C, show theeffect of purified NaPdf1 defensin protein against B. cinerea (FIG. 11),F. oxysporum (f. sp. dianthi, FIG. 12A) and F. oxysporum (f. sp.vasinfectum, FIGS. 12B and 12C), respectively. The results clearlyindicate the effectiveness of 20 μg/mL of NaPdf1 against all threefungal pathogens.

EXAMPLE 9 Production of Transgenic Tobacco Plants

(a) Construction of the Binary Plasmid

Primers FLOR1 and FLOR2, as shown below, were used to amplify the fullDNA coding sequence of NaPdf1 by conventional PCR, in order toincorporate a 5′ EcoRI and a 3′ XbaI restriction enzyme site forsubsequent cloning steps.

Primer FLOR1 (EcoRI-spacer-ATG-seq, 31 mer)5′ GGAATTCTAAACAATGGCTCGCTCCTTGTGC 3′ [SEQ ID NO:5] Primer FLOR2(XbaI-stop-seq, 29 mer) 5′ GCTCTAGATCAGTTATCCATTATCTCTTC 3′ [SEQ IDNO:6]

The resultant PCR product was directly sub-cloned into a TA vector(pCR2.1-TOPO, Invitrogen) and the sequence was verified by nucleotidesequencing. The NaPdf1 DNA was excised from the TA vector with EcoRI andXbaI restriction enzymes, gel purified and subcloned into the pFB98/06vector (obtained from Florigene Limited, Collingwood, Australia),previously treated with compatible restriction enzymes to createp35-PDF1. This construct (p35-PDF1) contains the defensin cDNA flankedby the ³⁵S cauliflower mosaic virus promoter (CaMV35S) and terminatorsequences. Construct p35-PDF1 was further digested with SphI restrictionenzyme, to remove the promoter-gene-terminator cassette, and gelpurified. The 3′ overhangs produced by SphI were blunt ended with T4 DNApolymerase and gel purified. At this stage, the cassette was insertedinto a binary vector, pGCP1988 (Florigene) or pBIN19 (Bevan et al.[1983; supra]), which had previously been treated with the blunt endcutter SmaI restriction enzyme. The resultant constructs were designated(i) pFL1 (contained in pCGP1988), which has a glean (surB) selectablemarker gene under the control of the CaMV35S promoter/terminator, and(ii) pHEX3 (contained in pBIN19) which has a kanamycin (nptII)selectable marker gene under the control of the nos promoter/terminator.Constructs pFL1 and pHEX3 have the defensin gene cassette in theconvergent and tandem orientation relative to their selectable markergenes, respectively. Schematics of constructs pFL1 and pHEX3 are shownin FIG. 13.

(b) Transformation of Agrobacterium Tumefaciens LBA4404

Electro-competent Agrobacterium tumefaciens (LBA4404) were prepared andtransformed with either pFL1 or pHEX3 by conventional electroporation ina Gene Pulser (registered trademark)/E. coli cuvettes with a 0.2 cmelectrode gap (Bio-Rad) and exposed to 1.8 kV, a capacitance of 25 μFDand a resistance of 600 ohms in a Gene Pulser (Bio-Rad). Theelectroporated cells were combined with 1 mL SOC medium and incubatedwith shaking at 28° C. for 3 h before 150 μL was withdrawn and platedout on LB agar supplemented with 20 μg/mL rifampicin for pFL1transformants or 20 μg/mL rifampicin and 50 μg/mL kanamycin for pHEX3transformants. The plates were incubated overnight at 28° C.

Transformants were selected by preparing plasmid DNA from randomlychosen colonies, performing diagnostic restriction digests and analysingfor inserts by agarose gel electrophoresis.

(c) Agrobacterium-Mediated Transformation of Tobacco

Seeds of N. tabacum cultivar Wisconscin 38 (W38) were surface sterilizedin 1% v/v sodium hypochlorite for 60 min followed by several washes insterile water. The sterilized seeds were sown onto MS medium (MS; 0.44%w/v Murashige and Skoog medium (ICN Biomedicals) [Plant Physiology 15:73–97, 1962], 3% w/v sucrose and 0.8% w/v Bacto agar, pH 5.8) andincubated in a temperature control cabinet for 5 weeks. A. tumefaciens(LBA4404) transformed with either the pFL1 or pHEX3 constructs weregrown for 2–3 days in 10 ml LB medium supplemented with the antibioticsrifampicin (20 μg/ml) or kanamycin (50 μg/ml) and rifampicin at 28° C.,respectively. The cells were collected by centrifugation (5 min,3,500×g), resuspended in 20 ml MS and incubated with freshly cut leafdisks (1 cm² squares) briefly. The disks were blotted onto sterile 3MMpaper before being transferred onto SIM agar (0.44% w/v Murashige andSkoog medium (ICN Biomedicals) [1962; supra], 3% w/v sucrose, 1.0 mg/LBAP, 0.5 mg/L indole acetic acid (IAA) and 0.8% w/v Bacto agar, pH 5.8)and incubated for 3 days at 25° C. in light. Control leaf disks weretreated similarly with MS alone. Following co-cultivation, calli weretransferred onto selective media to induce shoot formation. pFL1transformed calli were transferred to SIM agar supplemented with 1.5mg/L glean and 250 mg/L cefotaxamine while the pHEX3 transformed calliwere selected with 100 mg/L kanamycin and 250 mg/L cefotaxamine. Theregenerated shoots were dissected from the calli, briefly dipped in IBA(1 mg/mL) solution, and transferred onto root-inducing medium (RIM;0.44% w/v Murashige and Skoog medium [1962; supra], 3% w/v sucrose, 150mg/L timentin and 0.8% w/v Bacto agar), supplemented with either 1.5mg/L glean or 100 mg/L kanamycin for pFL1 or pHEX3, respectively andincubated as described previously. When adequate root growth wasestablished, plantlets were potted into soil and grown under standardglasshouse conditions.

(d) Detection of NaPdf1 in Transgenic Tobacco Leaves

Leaves (4^(th) or 5^(th) position) were cut at the petiole fromglasshouse grown plants. The tissue was frozen in liquid nitrogen andground to a fine powder with a mortar and pestle. The tissue was addedto extraction buffer: 50 mM Tris-HCl pH 8.0, 10 MM EDTA and 0.5 M NaCl(1 mL/g of fresh wet weight). The mixture was allowed to stand on icefor 15 min before clarification by centrifugation at 13,500×g for 15ml., 4° C. Protein concentrations were estimated by the method ofBradford (1976), reagents were from Bio-Rad and BSA was used as astandard. Total soluble leaf proteins (100 μg) were separated bySDS-PAGE using either 15% w/v or preformed 4–12% w/v polyacrylamidegradient gels (Novex) as described in Example 4(d), above. Followingelectrophoresis, proteins were either stained using coomassie blue ortransferred to nitrocellulose for immunodetection of NaPdf1 as describedin Example 4(e), above. Comparative results are shown in FIGS. 14A andB.

EXAMPLE 10 Insect Feeding Trials

(a) Source of Insects

Helicoverpa punctigera larvae came from a colony collected in Victoria,Australia and maintained in culture at La Trobe University, Melbourneand H. armigera were obtained from a colonies maintained at La TrobeUniversity, Melbourne or the Australian Cotton Research Institute, atthe Entomology Department of the Commonwealth Scientific and IndustrialResearch Organisation in Narrabri, NSW.

(b) Bioassays with Transgenic Tobacco Leaves

Three experiments were conducted with clonal material from transgenicplant, pFL1/W19 (transformed with the pFL1 construct) and transgenicplant, pHEX3.4 (transformed with the pHEX3 construct). An untransformedW38 plant was used as negative control.

In experiments 1 and 2, 31 and 40 newly hatched Helicoverpa punctigeralarvae were selected for each treatment, respectively. The larvae werereared in individual plastic cups with lids (Solo (registered trademark)plastic portion cups, 28 mL) containing 1.5% w/v Bacto agar and were fedleaf segments that were replaced either every 2–3 days or when more than75% had been consumed. The amount of leaf material was increased as thelarvae reached 5^(th) instar. Young leaves from non-flowering plantswere used in all bioassays. To avoid a wounding response, the leaveswere freshly excised from the petiole with a clean scalpel blade andwere divided into sections (2×2 cm) by careful dissection between themajor veins to minimize any wound response in the leaf sections. Thelarvae were kept in a controlled temperature room of 24±1° C., underlight. The weights of the larvae were measured every 2–3 days until day23 and the mean weight calculated. In experiment 3, 40 H. armigeralarvae were used under the same conditions described for the H.punctigera bioassays. Results are shown in FIGS. 15A–15D.

(c) Bioassays with Artificial Diet

To confirm that the insecticidal activity was due to the defensin andnot to upregulation of other endogenous defence molecules, a feedingtrial using artificial cotton leaf diet containing purified defensinfrom floral buds was performed. The 6 kDa cysteine rich proteinaseinhibitors (NAPI) from N. alata (Atkinson et al., Plant Cell 5: 203–213,1993) were used as positive control and casein was used as a negativecontrol. Feeding trials were conducted as described in Heath et al. (J.Insect Physiology 43: 833–842, 1997 except that the artificial diet wasbased on cotton leaves (Table 2). Thirty milligram of defensin waspurified from floral buds of N. alata and used in artificial diets attwo concentrations. H. armigera larvae fed on defensin at 0.3% (w/v)were 40% smaller than controls (FIG. 16) while larvae fed on NaPI at thesame concentration were about 60% smaller than controls.

EXAMPLE 11 Production of Transgenic Cotton Plants

(a) Agrobacterium-Mediated Transformation of Cotton

Seeds of Gossypium hirsutum cultivar Coker 315 were surface sterilizedin 2% v/v sodium hypochlorite for 60 min followed by several washes insterile water. The sterilized seed were sown onto half-strength MSmedium (MS; 0.22% w/v Murashige and Skoog salt mixture (Gibco BRL)[1962; supra], 0.2% Gelrite (Phyto Technology Laboratories), pH 5.8) andincubated at 30° C. in the dark for 7 days. A. tumefaciens (LBA4404)transformed with the pHEX3 construct was grown overnight in 25 ml LBmedium supplemented with the antibiotic kanamycin (50 μg/mL) at 28° C.The absorbance at 550 nm was measured and the cells were diluted to2×10⁸ cells per ml in MS liquid media (0.43% w/v Murashige and Skoogsalts [1962; supra], pH 5.8). Cotton hypocotyls were cut into 1.5–2 cmpieces and mixed briefly (0.5–3 min) in the diluted Agrobacteriumculture. The explants were blotted dry on sterile 3MM paper andtransferred to medium 1 (0.43% w/v Murashige and Skoog salt mixture(GibcoBRL) [1962; supra], 0.1% v/v Gamborg's B5 vitamin solution(Sigma), 0.1 g/L myo-inositol, 0.9 g/L MgCl₂, (hexahydrate), 1.9 g/Lpotassium nitrate, 0.2% w/v Gelrite, 3% w/v glucose, pH 5.8) overlayedwith sterile filter paper and incubated for 3 days at 26° C. underlights.

Following co-cultivation, explants were transferred to medium 2 (medium1 plus 0.1 mg/L kinetin, 0.1 mg/L 2,4-D, 500 mg/L carbenicillin, 35 mg/Lkanamycin) and maintained at 30° C. under low light. After 4 weeksexplants were transferred to medium 3 (medium 1 plus 500 mg/Lcarbenicillin, 35 mg/L kanamycin) and maintained at 30° C. under lowlight. Explants and callus were sub-cultured every 4 weeks on medium 3and maintained at 30° C. under low light. Embryos were excised from thetissue and germinated in medium 4 (1.2 mM CaCl₂2H₂O, 5.0 mM KNO₃, 2.0 mMMgSO₄7H₂O, 3.0 mM NH₄NO₃, 0.2 mM KH₂PO₄, 4 μM nicotinic acid, 4 μMpyridoxine HCl, 4 μM thiamine HCl, 30 μM H₃BO₃, 30 μM MnSO₄H₂O, 9 μMZnSO₄7H₂O, 1.5 μM KI, 0.9 pM Na₂MoO₄2H₂O, 0.03 μM CuSO₄5H₂O, 0.03 μMCoCl₂6H₂O, 0.5% w/v glucose, 0.3% w/v Gelrite, pH 5.5) and maintained at30° C. under high light.

Germinated embryos were then transferred to Magenta boxes containingmedium 5 (1.2 mM CaCl₂2H₂O, 40.0 mM KNO₃, 2.0 mM MgSO₄7H₂O, 15 mM NH₄Cl,0.2 mM KH₂PO₄, 4 μM nicotinic acid, 4 μM pyridoxine HCl, 4 μM thiamineHCl, 30 μM H₃BO₃, 30 μM MnSO₄H₂O, 9 μM ZnSO₄7H₂O, 1.5 μM KI, 0.9 μMNa₂MoO₄2H₂O, 0.03 μM CuSO₄5H₂O, 0.03 μM CoCl₂6H₂O, 2.0% w/v sucrose,0.2% w/v Gelrite, pH 5.5) and maintained at 30° C. under high light.Once a plant has formed a good root system and produced several newleaves it was transferred to soil in pots and acelimatised in a growthcabinet at 28° C. and then grown in a glasshouse at (27–29° C. day,20–24° C. night).

(b) Detection of NaPdf1 in Transgenic Cotton

Leaves (first position, 3–4 cm in diameter) were excised from plantsgrown either in the growth cabinet or in the glasshouse. The tissue (100mg) was frozen in liquid nitrogen and ground to a fine powder with amortar and pestle. The powder was added to 2× sample buffer (300 μl,Novex NUPAGE LDS sample buffer, 10% v/v β-mercaptoethanol), vortexed for30 sec, boiled for 5 min and then centrifuged at 14,000 rpm for 10 minand the supernatant retained. Total soluble leaf extracts were separatedby SDS-PAGE on preformed 4–12% w/v polyacrylamide gradient gels (Novex,NuPAGE bis-tris, MES buffer) for 35 min at 200V in a Novex X Cell IImini-cell electrophoresis apparatus. Prestained molecular weight markers(Novex SeeBlue) were included as a standard. Proteins were transferredto nitrocellulose membrane (Micron Separations Inc. 0.22 micron poresize) for 60 min at 30V using the Novex X Cell II mini-cellelectrophoresis apparatus in NuPAGE transfer buffer with 10% v/vmethanol. After transfer, membranes were incubated for 1 min inisopropanol, followed by a 5 min wash in TBS.

The membrane was blocked for 1 h in 3% w/v BSA at RT followed byincubation with primary antibody overnight at RT (1:2500 dilution inTBS). The membrane was washed 5×10 min in TBST before incubation withgoat anti-rabbit IgG conjugated to horseradish peroxidase for 60 min atRT (Pierce, 1:100,000 dilution in TBS). Five further 10 min TBST washeswere performed before the membrane was incubated with the SuperSignalWest Pico Chemiluminescent substrate (Pierce) according to theManufacturer's instructions. Membranes were exposed to ECL Hyperfilm(Amersham Pharmacia Biotech). Results are shown in FIG. 14C.

EXAMPLE 12 Insect Feeding Trial with Transgenic Cotton

One experiment was conducted with two independently transformed T2generation transgenic cotton lines. The plants were produced by selfingthe primary transgenic lines CT35.9.4 and CT35.125.1 (both transformedwith the pHEX3 construct) and consisted of a mixture of homozygous andhemizygous plants. Parent untransformed Coker 315 plants were used as anegative control.

Twenty newly hatched H. armigera larvae were selected. The larvae werereared in individual plastic cups with lids (Solo (registered trademark)plastic portion cups, 28 ml) containing 1.5% Bacto agar and were fedleaf segments that were replaced either every 2–3 days or when more than75% had been consumed. Young leaves from non-flowering plants were usedin the bioassay. The larvae were kept in a controlled temperature roomat 25±1° C., under light. The number of dead larvae was recorded on days4, 6 and 8. The weight of each surviving larvae was measured at day 8and the mean weight calculated. Results are shown in FIG. 17.

EXAMPLE 13 Production of Insect-resistant Plants

(a) Molecular Cloning of Genes from a Range of Species

Amino acid sequences of any one of the sequences set forth in FIG. 10(SEQ ID NO:25 to SEQ ID NO:49) are used to design suitableoligonucleotide primers for use in screening cDNA or genomic DNAlibraries, as appropriate. Using, for example PCR, corresponding fullnucleotide coding sequences are cloned as, for example, described inExamples 4(a) and 9(a), above. Expression cassettes are then insertedinto desired vectors such as, for example, pBIN19 or pCGP1988, use ofwhich is described in Example 9(a).

(b) Transformation and Regeneration of Insect-resistant Plants

Using standard techniques known and available to those skilled in theart, selected plant material of target plant species is transformed withone or more vectors comprising the expression cassettes carrying theanti-insect sequences from the corresponding species. Suitabletransformation methods include, but are not limited to, theAgrobacterium-mediated transformation protocol set forth in Example9(b), 9(c) and 9(d), modified as necessary for particular plant species.Other means of transformation of particular plant species are well knownand include, for example, biolistic transformation procedures.

Following regeneration, plants are assayed for resistance to attack bycommon plant pests including insects.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. An isolated nucleic acid molecule comprising a sequence ofnucleotides encoding or complementary to a sequence encoding apolypeptide comprising the amino acid sequence as set forth in SEQ IDNO:8, wherein said polypeptide has activity against a plant pest,wherein said nucleic acid molecule further comprises a sequence ofnucleotides encoding a C-terminal amino acid sequence and/or a sequenceof nucleotides encoding an N-terminal signal sequence.
 2. The isolatednucleic acid molecule of claim 1, wherein said nucleic acid moleculefurther comprises a sequence of nucleotides encoding a C-terminal aminoacid sequence and wherein the C-terminal amino acid sequence comprisesthe sequence as set forth in SEQ ID NO:12.
 3. The isolated nucleic acidmolecule of claim 1 or 2, wherein said nucleic acid molecule furthercomprises a sequence of nucleotides encoding an N-terminal signalsequence and wherein the N-terminal signal sequence comprises thesequence as set forth in SEQ ID NO:10.
 4. The isolated nucleic acidmolecule of claim 1, wherein the nucleotide sequence encodes the aminoacid sequence set forth in SEQ ID NO:18.
 5. The isolated nucleic acidmolecule of claim 1, comprising the nucleotide sequence as set forth inSEQ ID NO:7.
 6. The isolated nucleic acid molecule of claim 2, whereinthe C-terminal amino acid sequence is encoded by the nucleotide sequenceas set forth in SEQ ID NO:11.
 7. The isolated nucleic acid molecule ofclaim 3, wherein the N-terminal signal sequence is encoded by thenucleotide sequence as set forth in SEQ ID NO:9.
 8. The isolated nucleicacid molecule of claim 4, comprising the nucleotide sequence as setforth in SEQ ID NO:17.
 9. A genetically modified plant cell, tissue,organ or whole plant which expresses the nucleic acid molecule ofclaim
 1. 10. The plant cell, tissue, organ or whole plant of claim 9,wherein the plant is a cotton plant.
 11. A progeny of the plant of claim9 or 10, wherein said progeny expresses the nucleic acid molecule.
 12. Amethod for generating a plant with increased resistance to a plant pest,said method comprising introducing into a genome of a plant cell anexpressible form of the nucleic acid molecule of claim 1, 2 or 4 andregenerating a plant from said cell.
 13. The method of claim 12 whereinthe plant is a cotton plant.
 14. The method of claim 12 wherein saidnucleic acid is expressed in epidermal layers of petals and sepals,cortical cells of a style and/or the connective tissue of an anther.